PrP-like gene

ABSTRACT

The present invention provides nucleic acids encoding the Doppel (“Dpl”) protein, Dpl peptides, and assays utilizing the Dpl nucleic acids and/or peptides. In related aspects the invention features expression vectors and host cells comprising nucleic acids that encode a human Dpl polypeptide. The present invention also relates to antibodies that bind specifically to a human Dpl polypeptide, methods for producing human Dpl polypeptides, methods for identifying cells expressing Dpl, methods for using the Dpl gene and the Dpl polypeptide to alter cellular function and prion infectivity in culture or in vivo, and identification of individuals at risk for prion disorders by detecting alteration in Dpl coding and regulatory sequences and Dpl expression levels.

GOVERNMENT RIGHTS

[0001] The United States Government may have certain rights in thisapplication pursuant to Grant XXXX, etc.

FIELD OF THE INVENTION

[0002] This invention relates to nucleic acids, proteins encoded by suchnucleic acids, and assays involving use of the these nucleic acidsand/or proteins.

BACKGROUND OF THE INVENTION

[0003] Prions are infectious pathogens that cause central nervous systemspongiform encephalopathies in humans and animals. Prions are distinctfrom bacteria, viruses and viroids. The predominant hypothesis atpresent is that no nucleic acid component is necessary for infectivityof prion protein. Further, a prion which infects one species of animal(e.g., a human) will not readily infect another (e.g., a mouse).

[0004] A major step in the study of prions and the diseases that theycause was the discovery and purification of a protein designated prionprotein (“PrP”) [Bolton et al., Science 218:1309-11 (1982); Prusiner etal., Biochemistry 21:6942-50 (1982); McKinley et al., Cell 35:57-62(1983)]. Complete prion protein-encoding genes have since been cloned,sequenced and expressed in transgenic animals. PrP^(c) is encoded by asingle-copy host gene [Basler et al., Cell 46:417-28 (1986)] and isnormally found at the outer surface of neurons. A leading hypothesis isthat prion diseases result from conversion of PrP^(c) into a modifiedform called PrP^(Sc).

[0005] It appears that the scrapie isoform of the prion protein(PrP^(Sc)) is necessary for both the transmission and pathogenesis ofthe transmissible neurodegenerative diseases of animals and humans. SeePrusiner, S. B., “Molecular biology of prion disease,” Science252:1515-1522 (1991). The most common prion diseases of animals arescrapie of sheep and goats and bovine spongiform encephalopathy (BSE) ofcattle [Wilesmith, J. and Wells, Microbiol. Immunol. 172:21-38 (1991)].Four prion diseases of humans have been identified: (1) kuru, (2)Creutzfeldt-Jakob Disease (CJD), (3) Gerstmann-Strassler-ScheinkerDisease (GSS), and (4) fatal familial insomnia (FFI) [Gajdusek, D. C.,Science 197:943-960 (1977); Medori et al., N. Engl. J. Med. 326:444-449(1992)]. The presentation of human prion diseases as sporadic, geneticand infectious illnesses initially posed a conundrum which has beenexplained by the cellular genetic origin of PrP.

[0006] Most CJD cases are sporadic, but about 10-15% are inherited asautosomal dominant disorders that are caused by mutations in the humanPrP gene [Hsiao et al., Neurology 40:1820-1827 (1990); Goldfarb et al.,Science 258:806-808 (1992); Kitamoto et al., Proc. R. Soc. Lond.343:391-398. Iatrogenic CJD has been caused by human growth hormonederived from cadaveric pituitaries as well as dura mater grafts [Brownet al., Lancet 340:24-27 (1992)]. Despite numerous attempts to link CJDto an infectious source such as the consumption of scrapie infectedsheep meat, none has been identified to date [Harries-Jones et al., J.Neurol. Neurosurg. Psychiatry 51:1113-1119 (1988)] except in cases ofiatrogenically induced disease. On the other hand, kuru, which for manydecades devastated the Fore and neighboring tribes of the New Guineahighlands, is believed to have been spread by infection duringritualistic cannibalism [Alpers, M. P., Slow Transmissible Diseases ofthe Nervous System, Vol. 1, S. B. Prusiner and W. J. Hadlow, eds. (NewYork: Academic Press), pp. 66-90 (1979)].

[0007] The initial transmission of CJD to experimental primates has arich history beginning with William Hadlow's recognition of thesimilarity between kuru and scrapie. In 1959, Hadlow suggested thatextracts prepared from patients dying of kuru be inoculated intononhuman primates and that the animals be observed for disease that waspredicted to occur after a prolonged incubation period [Hadlow, W. J.,Lancet 2:289-290 (1959)]. Seven years later, Gajdusek, Gibbs and Alpersdemonstrated the transmissibility of kuru to chimpanzees afterincubation periods ranging form 18 to 21 months [Gajdusek et al., Nature209:794-796 (1966)]. The similarity of the neuropathology of kuru withthat of CJD [Klatzo et al., Lab Invest. 8:799-847 (1959)] promptedsimilar experiments with chimpanzees and transmissions of disease werereported in 1968 [Gibbs, Jr. et al., Science 161:388-389 (1968)]. Overthe last 25 years, about 300 cases of CJD, kuru and GSS have beentransmitted to a variety of apes and monkeys.

[0008] The expense, scarcity and often perceived inhumanity of suchexperiments have restricted this work and thus limited the accumulationof knowledge. While the most reliable transmission data has been said toemanate from studies using nonhuman primates, some cases of human priondisease have been transmitted to rodents but apparently with lessregularity [Gibbs, Jr. et al., Slow Transmissible Diseases of theNervous System, Vol. 2, S. B. Prusiner and W. J. Hadlow, eds. (New York:Academic Press), pp. 87-110 (1979); Tateishi et al., Prion Diseases ofHumans and Animals, Prusiner et al., eds. (London: Ellis Horwood), pp.129-134 (1992)].

[0009] The importance of understanding the conversion of PrP^(c) intoPrP^(Sc) has been heightened by the possibility that bovine prions havebeen transmitted to humans who developed variant Creutzfeldt-Jakobdisease (vCJD), G. Chazot, et al., Lancet 347:1181 (1996); R. G. Will,et al., Lancet 347:921-925 (1996). Earlier studies had shown that theN-terminus of PrP^(Sc) could be truncated without loss of scrapieinfectivity, S. B. Prusiner, et al., Biochemistry 21:6942-6950 (1982);S. B. Prusiner, et al., Cell 38:127-134 (1984) and correspondingly, thetruncation of the N-terminus of PrP^(Sc) still allowed its conversioninto PrP^(Sc) M. Rogers, et al., Proc. Natl. Acad. Sci. USA 90:3182-3186(1993).

[0010] Recent studies have advanced the ability to visualize thestructural transition of PrP^(c) to PrP^(Sc) at a molecular level. Forexample, the N-terminal portion is relatively unstructured and flexible,but assists in stabilizing structural elements within the C-terminalportion. D. G. Donne et al., Proc. Natl. Acad. Sci. USA 94:13452-13457(1997). Furthermore, immunological studies have demonstrated thatN-terminal epitopes are cryptic in PrP^(Sc), supporting the idea thatthis region undergoes profound conformational change during prionpropagation. Peretz et al., J. Mol. Biol. 273:614-622 (1997).

[0011] Despite these advances, the understanding of the structuralbiology of the pathogenic conversion process remains incomplete in manyways. For example, it is unknown exactly which structural regions ofPrP^(c) are necessary or sufficient for conformational change to occur.It is also unknown which regions of PrP^(Sc) are necessary or sufficientfor infectivity. Evidence indicates that prion strain phenomena andspecies barriers are encoded by alternative PrP conformations, but theprecise structural determinants of these conformations have not yet beenprecisely identified. Telling et al. Science 274:2079-2082 (1996);Billeter, et al., Proc. Natl. Acad. Sci. USA 94:7281-7285 (1997). Recentstudies have identified four residues of mouse PrP (MoPrP) that appearto interact with protein X, a putative factor postulated to facilitatethe conformational change from PrP^(c) to PrP^(Sc). Telling, et. al.Cell 83:79-90 (1995). All four amino acids come together to form theputative protein X binding site in the tertiary structure of recombinantPrIP 90-231 and PrIP 29-231. D. G. Donne et al., Proc. Natl. Acad. Sci.USA 94:13452-13457 (1997); T. L. James et al., Proc. Natl. Acad. Sci.USA 94:10086-10091 (1997). However, despite several reports of proteinswhich bind PrP^(c), the identity of protein X remains elusive. Finally,although the structures of refolded, recombinant PrP molecules mayresemble PrP^(c), a structural solution for PrP^(Sc) remains lacking.

[0012] One strategy for determining PrP function is through theidentification of proteins similar to PrP, and the elucidation of thefunction of such proteins. The identification and study of prion-relatedgenes may offer insight into the general biology and progression ofneurodegenerative disorders, as well as offering insight into themechanistic alterations that result in prion-mediated disorders. Thereis thus a need in the art for the identification and study of genesencoding proteins with similar structure and/or function.

SUMMARY OF THE INVENTION

[0013] The present invention provides nucleic acids encoding the Doppel(“Dpl”) protein, Dpl peptides, and assays utilizing the Dpl nucleicacids and/or peptides. In a particular aspect, the Dpl protein isencoded by the nucleotide sequence of SEQ ID NO:1 and degeneratesequences thereof. In addition, the invention features isolated nucleicacid sequences comprising a Dpl promoter, as well as nucleic acidsequences that hybridize under stringent conditions to SEQ ID NO:1. Inrelated aspects the invention features expression vectors and host cellscomprising nucleic acids that encode a human Dpl polypeptide. Thepresent invention also relates to antibodies that bind specifically to ahuman Dpl polypeptide, methods for producing human Dpl polypeptides,methods for identifying cells expressing Dpl, methods for using the Dplgene and the Dpl polypeptide to alter cellular function and prioninfectivity in culture or in vivo, and identification of individuals atrisk for degenerative disorders by detecting alteration in Dpl codingand regulatory sequences and Dpl expression levels.

[0014] A primary object of the invention is to provide an isolated humanDpl polypeptide-encoding nucleic acids for use in expression of humanDpl (e.g, in a recombinant host cell) and for use in, for example,identification of human Dpl polypeptide binding compounds (especiallythose compounds that affect human Dpl polypeptide-mediated activity,which compounds can be used to modulate Dpl activity).

[0015] Another object of the invention is to provide an isolated humanDpl polypeptide-encoding nucleic acid for use in generation of non-humantransgenic animal models for Dpl gene function, particularly “knock-in”Dpl non-human transgenic animals characterized by excess or ectopicexpression of the Dpl gene.

[0016] In particular, Dpl appears to have a synergistic interaction withthe products of the PrP locus, and mutations in the Dpl locus decreasesthe incubation period of animals infected with the scrapie form of PrP,PrP^(Sc). DNA encoding Dpl is also disclosed for use of the DNAexpression product in an assay to modulate the incubation time neededfor prion-mediated disorders, as well as to identify compounds thatmodulate the incubation time necessary for the measurable onset ofsymptoms of prion infection.

[0017] The invention also features DNA sequences that hybridize understringent conditions to DNA encoding Dpl, or to DNA complementary to DNAencoding Doppel. Such DNA sequences are preferably at least 25nucleotides in length, more preferably 50 nucleotides in length. Peptidefragments of Dpl, e.g. those useful in assays, are preferably at least10 amino acids in length, more preferably at least 30 amino acids inlength.

[0018] The invention also features chimeric Dpl genes having sequencesfrom both a host animal and a genetically diverse animal, and transgenicanimals containing such genes.

[0019] An object of the invention is to provide a nucleotide sequenceencoding a novel Dpl protein and its functional equivalents.

[0020] Another object is to provide a cell line genetically engineeredto express the nucleotide sequence encoding Dpl.

[0021] Another object is to provide an antibody which selectively bindsto the Dpl protein or peptides thereof.

[0022] Another object is to provide a method whereby a compound orlibrary of compounds can be assayed for their ability to activate orblock the activity of the protein expressed by the Dpl nucleotidesequence.

[0023] An advantage of the present invention is that mutations thatincrease Dpl activity and/or expression accelerate prion infection, thusallowing an earlier identification of prion infectivity from an inoculumin animal assays, e.g. in transgenic HuPrP mouse assays.

[0024] An aspect of the invention is transgenic mice expressing (1)human Dpl; (2) human Dpl and human PrP; (3) human/mouse chimeric genesfor Dpl and PrP; (4) bovine Dpl; (5) bovine Dpl and bovine PrP; (6)bovine/mouse chimeric genes for Dpl and PrP; (7) combination of (1-6)with an ablated endogeneous Dpl and/or PrP gene; (7) sheep Dpl; (8) goatDpl; (9) elk Dpl; and (10) deer Dpl. Transgenic mice that may be used inthe present invention are described in U.S. Pat. Nos. 5,565,186 issuedOct. 15, 1996, 5,763,740 issued Jun. 9, 1998, 5,789,655 issued Aug. 4,1998, and 5,792,901, issued Aug. 11, 1998, which are incorporated byreference herein.

[0025] These and other objects, advantages and features of the presentinvention will become apparent to those skilled in the art upon readingthe disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026]FIG. 1A is a schematic diagram illustrating the genomic structureand coding regions of the Dpl (PrnD) gene locus. FIG. 1B illustrates thesplice variants of Dpl, as well as identified ESTs in this region.

[0027]FIG. 2 is the nucleotide and corresponding amino acid sequence ofthe mouse Dpl gene.

[0028]FIG. 3 is a schematic diagram illustrating the alternative splicedonor sites of Exon 1.

[0029]FIG. 4 is a structural overview of Prn gene region, with the mostabundant forms Prnp and PrnD spliced mRNAs (thin lines) shown at the topof the figure. Chimeric cDNAs are shown in bold lines, with thepositions of oligonucleotide primers (arrows) used for RT-PCR shownabove the chromosomal gene.

[0030]FIG. 5 is a schematic diagram illustrating the structure of DplcDNA varieties Prnp exon 2 and PrnD exon 2 sequences common to all cDNAsare shown by bold cross-hatch and open boxes, respectively.

[0031]FIG. 6 is a schematic diagram illustrating the structures of PrPgene disruptions in four lines of Prnp^(0/0) mice. The large blackarrows indicate the alleles which delete the exon 3 splice acceptor andare associated with the development of a late-onset ataxia. A smallvertical arrow indicates the position of the exon 3 splice acceptordeleted in the Ngsk and Rcm alleles. The open box is the PrP codingregion and the gray box is the PrP UTR. Enzymes are as follows: E, EcoRI; X, Xba I; K, Kpn I.

[0032]FIG. 7 and 8 illustrate multiple sequence alignment of human,mouse and rat Dpl with representative PrP molecules showing the regionsof identity and conservation.

[0033]FIG. 9 is a schematic of the Dpl and PrP protein structure showingthe positions of the predicted features of each. Three alpha helicesfound in PrP are shown as grey boxes with A, B and C representing thethree predicted Dpl helices. The white box with the + symbols indicate acluster of basic residues.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0034] Before the present DNA molecule, proteins, and methods of use aredescribed, it is to be understood that this invention is not limited toparticular molecules described, as such may, of course, vary. It is alsoto be understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present invention will be limited onlyby the appended claims.

[0035] Unless defined otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any methodsand materials similar or equivalent to those described herein can beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

[0036] The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DEFINITIONS

[0037] The term “nucleic acid” as used herein refers to anoligonucleotide, nucleotide, and fragments or portions thereof, as wellas to peptide nucleic acids (PNA), fragments, portions or antisensemolecules thereof, and to DNA or RNA of genomic or synthetic originwhich can be single- or double-stranded, and represent the sense orantisense strand. Where “nucleic acid”is used to refer to a specificnucleic acid sequence (e.g. a Dpl polypeptide-encoding nucleic acid),“nucleic acid”is meant to encompass nucleic acids that encode apolypeptide that is functionally equivalent to the recited polypeptide,e.g., nucleic acids that are degenerate variants, or nucleic acids thatencode biologically active variants or fragments of the recitedpolypeptide. Similarly, “polypeptide” as used herein refers to anoligopeptide, peptide, or protein. Where “polypeptide” is recited hereinto refer to an amino acid sequence of a naturally-occurring proteinmolecule, “polypeptide” and like terms are not meant to limit the aminoacid sequence to the complete, native amino acid sequence associatedwith the recited protein molecule.

[0038] By “antisense nucleic acid”is mean a nucleic acid having anucleotide sequence complementary to a given nucleic acid sequence (e.g,a nucleic acid sequence encoding a Dpl polypeptide) including nucleicacid sequences associated with the transcription or translation of thegiven nucleic acid sequence (e.g., a promoter of a nucleic acid encodinga Dpl polypeptide), where the antisense nucleic acid is capable ofhybridizing to a Dpl polypeptide-encoding nucleic acid sequence. Ofparticular interest are antisense nucleic acids capable of inhibitingtranscription and/or splicing and/or translation of a Dpl-encodingnucleic acid either in vitro or in vivo.

[0039] “Peptide nucleic acid”as used herein refers to a molecule whichcomprises an oligomer to which an amino acid residue, such as lysine,and an amino group have been added. These small molecules, alsodesignated anti-gene agents, stop transcript elongation by binding totheir complementary (template) strand of nucleic acid (Nielsen et al.,Anticancer Drug Des 8:53-63 (1993)).

[0040] By a “polypeptide” is meant any chain of amino acids, regardlessof length or post-translational modification (e.g., glycosylation).

[0041] As used herein, “Dpl polypeptide” refers to an amino acidsequence of a recombinant or nonrecombinant polypeptide having an aminoacid sequence of i) a native Dpl polypeptide, ii) a biologically activefragment of a Dpl polypeptide, iii) biologically active polypeptideanalogs of a Dpl polypeptide, or iv) a biologically active variant of aDpl polypeptide. Dpl polypeptides of the invention can be obtained fromany species, e.g, mammalian or non-mammalian (e.g., reptiles,amphibians, avian (e.g., chicken)), particularly mammalian, includinghuman, rodent (e.g., murine or rat), bovine, ovine, porcine, murine, orequine, preferably rat or human, from any source whether natural,synthetic, semi-synthetic or recombinant. “Human Dpl polypeptide” refersto the amino acid sequences of isolated human Dpl polypeptide obtainedfrom a human, and is meant to include all naturally-occurring allelicvariants, and is not meant to limit the amino acid sequence to thecomplete, native amino acid sequence associated with the recited proteinmolecule.

[0042] As used herein, “antigenic amino acid sequence” means an aminoacid sequence that, either alone or in association with a carriermolecule, can elicit an antibody response in a mammal.

[0043] A “variant” of a human Dpl polypeptide is defined as an aminoacid sequence that is altered by one or more amino acids. The variantcan have “conservative” changes, wherein a substituted amino acid hassimilar structural or chemical properties, e.g., replacement of leucinewith isoleucine. More rarely, a variant can have “nonconservative”changes, e.g., replacement of a glycine with a tryptophan. Similar minorvariations can also include amino acid deletions or insertions, or both.Guidance in determining which and how many amino acid residues may besubstituted, inserted or deleted without abolishing biological orimmunological activity can be found using computer programs well knownin the art, for example, DNAStar software.

[0044] A “deletion” is defined as a change in either amino acid ornucleotide sequence in which one or more amino acid or nucleotideresidues, respectively, are absent as compared to an amino acid sequenceor nucleotide sequence of a naturally occurring Dpl polypeptide.

[0045] An “insertion” or “addition” is that change in an amino acid ornucleotide sequence which has resulted in the addition of one or moreamino acid or nucleotide residues, respectively, as compared to an aminoacid sequence or nucleotide sequence of a naturally occurring Dplpolypeptide.

[0046] A “substitution” results from the replacement of one or moreamino acids or nucleotides by different amino acids or nucleotides,respectively as compared to an amino acid sequence or nucleotidesequence of a naturally occurring Dpl polypeptide.

[0047] The term “biologically active” refers to human Dpl polypeptidehaving structural, regulatory, or biochemical functions of a naturallyoccurring Dpl polypeptide. Likewise, “immunologically active” definesthe capability of the natural, recombinant or synthetic human Dplpolypeptide, or any oligopeptide thereof, to induce a specific immuneresponse in appropriate animals or cells and to bind with specificantibodies.

[0048] The term “derivative” as used herein refers to the chemicalmodification of a nucleic acid encoding a human Dpl polypeptide or theencoded human Dpl polypeptide. Illustrative of such modifications wouldbe replacement of hydrogen by an alkyl, acyl, or amino group. A nucleicacid derivative would encode a polypeptide which retains essentialbiological characteristics of a natural Dpl polypeptide.

[0049] As used herein the term “isolated” is meant to describe acompound of interest (e.g., either a nucleic acid or a polypeptide) thatis in an environment different from that in which the compound naturallyoccurs. “Isolated” is meant to include compounds that are within samplesthat are substantially enriched for the compound of interest and/or inwhich the compound of interest is partially or substantially purified.

[0050] As used herein, the term “substantially purified” refers to acompound (e.g, either a nucleic acid or a polypeptide) that is removedfrom its natural environment and is at least 60% free, preferably 75%free, and most preferably 90% free from other components with which itis naturally associated.

[0051] “Stringency” typically occurs in a range from about Tm−5 ° C. (5° C. below the Tm of the probe) to about 20 ° C. to 25 ° C. below Tm. Aswill be understood by those of skill in the art, a hybridizationstringency can be manipulated to identify or detect identical nucleicacid sequences or to identify or detect similar or related nucleic acidsequences.

[0052] The term “hybridization” as used herein shall include “anyprocess by which a strand of nucleic acid joins with a complementarystrand through base pairing” (Coombs, Dictionary of Biotechnology,Stockton Press, New York N.Y. (1994)). Amplification as carried out inthe polymerase chain reaction technologies is described in Dieffenbachet al., PCR Primer, a Laboratory Manual, Cold Spring Harbor Press,Plainview N.Y. (1995).

[0053] By “transformation” is meant a permanent or transient geneticchange, preferably a permanent genetic change, induced in a cellfollowing incorporation of new DNA (i.e., DNA exogenous to the cell).Genetic change can be accomplished either by incorporation of the newDNA into the genome of the host cell, or by transient or stablemaintenance of the new DNA as an episomal element. Where the cell is amammalian cell, a permanent genetic change is generally achieved byintroduction of the DNA into the genome of the cell.

[0054] By “construct” is meant a recombinant nucleic acid, generallyrecombinant DNA, that has been generated for the purpose of theexpression of a specific nucleotide sequence(s), or is to be used in theconstruction of other recombinant nucleotide sequences.

[0055] By “operably linked” is meant that a DNA sequence and aregulatory sequence(s) are connected in such a way as to permit geneexpression when the appropriate molecules (e.g., transcriptionalactivator proteins) are bound to the regulatory sequence(s).

[0056] By “operatively inserted” is meant that a nucleotide sequence ofinterest is positioned adjacent a nucleotide sequence that directstranscription and translation of the introduced nucleotide sequence ofinterest (i.e., facilitates the production of, e.g., a polypeptideencoded by a Dpl sequence).

[0057] By “Dpl associated disorder” is meant a physiological conditionor disease associated with altered Dpl function (e.g., due to aberrantDpl expression or a defect in Dpl expression or in the Dpl protein).Such Dpl associated disorders can include, but are not necessarilylimited to, prion-like disorders or other similar disorders that involveneurotoxicity and/or neurodegeneration.

[0058] The term “transgene” is used herein to describe genetic materialwhich has been or is about to be artificially inserted into the genomeof a mammalian, particularly a mammalian cell of a living animal.

[0059] By “transgenic organism” is meant a non-human organism (e.g.,single-cell organisms (e.g., yeast), mammal, non-mammal (e.g., nematodeor Drosophila)) having a non-endogenous (i.e., heterologous) nucleicacid sequence present as an extrachromosomal element in a portion of itscells or stably integrated into its germ line DNA.

[0060] By “transgenic animal” is meant a non-human animal, usually amammal, having a non-endogenous (i.e., heterologous) nucleic acidsequence present as an extrachromosomal element in a portion of itscells or stably integrated into its germ line DNA (i.e., in the genomicsequence of most or all of its cells). Heterologous nucleic acid isintroduced into the germ line of such transgenic animals by geneticmanipulation of, for example, embryos or embryonic stem cells of thehost animal. A mouse is a preferred transgenic animal.

[0061] A “knock-out” of a target gene means an alteration in thesequence of the gene that results in a decrease of function of thetarget gene, preferably such that target gene expression is undetectableor insignificant. A knock-out of a Dpl gene means that function of theDpl gene has been substantially decreased so that Dpl expression is notdetectable or only present at insignificant levels. “knock-out”transgenics of the invention can be transgenic animals having aheterozygous knock-out of the Dpl gene or a homozygous knock-out of theDpl gene. “Knock-outs” also include conditional knock-outs, wherealteration of the target gene can occur upon, for example, exposure ofthe animal to a substance that promotes target gene alteration,introduction of an enzyme that promotes recombination at the target genesite (e.g., Cre in the Cre-lox system), or other method for directingthe target gene alteration postnatally.

[0062] A “knock-in” of a target gene means an alteration in a host cellgenome that results in altered expression (e.g., increased (includingectopic) or decreased expression) of the target gene, e.g., byintroduction of an additional copy of the target gene, or by operativelyinserting a regulatory sequence that provides for enhanced expression ofan endogenous copy of the target gene. “knock-in” transgenics of theinvention can be transgenic animals having a heterozygous knock-in ofthe Dpl gene or a homozygous knock-in of the Dpl gene. “Knock-ins” alsoencompass conditional knock-ins.

[0063] The term “Prnp^(0/0) or Prnp-Ab1 ” refers to a transgenic animalwhich has its PrP gene ablated with the “^(0/0) ” indicating that bothalleles are ablated whereas o/+ indicates only one is ablated.Specifically, the animal being referred to is generally a transgenicmouse which has its PrP gene ablated i.e., a PrP knockout mouse. In thatthe PrP gene is disrupted, no mouse PrP protein is expressed.

[0064] The term “prion” shall mean an infectious particle known to causediseases (spongiform encephalopathies) in animals including cows andhumans. The term “prion” is a contraction of the words “protein” and“infection” and the particles are comprised largely if not exclusivelyof PrP^(Sc) molecules encoded by a PrP gene. Prions are distinct frombacteria, viruses and viroids. Known prions include those which infectanimals to cause scrapie, a transmissible, degenerative disease of thenervous system of sheep and goats as well as bovine spongiformencephalopathies (BSE) or “mad cow” disease and feline spongiformencephalopathies of cats. Four prion diseases known to affect humans are(1) kuru, (2) Creutzfeldt-Jakob Disease (CJD), (3)Gerstmann-Sträussler-Scheinker Disease (GSS), and (4) fatal familialinsomnia (FFI). As used herein prion includes all forms of prionscausing all or any of these diseases or others in any animals used C andin particular in humans, cows and other domesticated farm animals.

[0065] The terms “PrP gene” and “Prnp gene” are used interchangeablyherein to describe genetic material which expresses proteins (forexample those shown in FIGS. 3-5 of U.S. Pat. No. 5,565,186 issued Oct.15, 1996) and polymorphisms and mutations such as those listed hereinunder the subheading “Pathogenic Mutations and Polymorphisms.” The PrPgene can be from any animal including the “host” and “test” animalsdescribed herein and any and all polymorphisms and mutations thereof, itbeing recognized that the terms include other such PrP genes that areyet to be discovered.

[0066] The term “artificial PrP gene” is used herein to encompass theterm “chimeric PrP gene” as well as other recombinantly constructedgenes which when included in the genome of a host animal (e.g., a mouse)will render the mammal susceptible to infection from prions whichnaturally only infect a genetically diverse test mammal, e.g., human,bovine or ovine. In general, an artificial gene will include the codonsequence of the PrP gene of the mammal being genetically altered withone or more (but not all, and generally less than 40) codons of thenatural sequence being replaced with a different codon C preferably acorresponding codon of a genetically diverse mammal (such as a human).The genetically altered mammal being used to assay samples for prionswhich only infect the genetically diverse mammal. Examples of artificialgenes are mouse PrP genes encoding the sequence as shown in FIGS. 3, 4and 5 of U.S. Pat. No. 5,565,186 with one or more different replacementcodons selected from the codons shown in these Figures for humans, cowsand sheep replacing mouse codons at the same position, with the provisothat not all the mouse codons are replaced with differing human, cow orsheep codons. Artificial PrP genes of the invention can include not onlycodons of genetically diverse animals but may include codons and codonsequences associated with genetic prion diseases such as CJD and codonsand sequences not associated with any native PrP gene but which, wheninserted into an animal render the animal susceptible to infection withprions which would normally only infect a genetically diverse animal.

[0067] The terms “chimeric gene,” “chimeric PrP gene” and the like areused interchangeably herein to mean an artificially constructed genecontaining the codons of a host animal such as a mouse with one or moreof the codons being replaced with corresponding codons from agenetically diverse test animal such as a human, cow or sheep. In onespecific example the chimeric gene is comprised of the starting andterminating sequence (i.e., N- and C- terminal codons) of a PrP gene ofa mammal of a host species (e.g. a mouse) and also containing anucleotide sequence of a corresponding portion of a PrP gene of a testmammal of a second species (e.g. a human). A chimeric gene will, wheninserted into the genome of a mammal of the host species, render themammal susceptible to infection with prions which normally infect onlymammals of the second species. The preferred chimeric gene disclosedherein is MHu2M which contains the starting and terminating sequence ofa mouse PrP gene and a non-terminal sequence region which is replacedwith a corresponding human sequence which differs from a mouse PrP genein a manner such that the protein expressed thereby differs at nineresidues.

[0068] The terms “host animal” and “host mammal” are used to describeanimals which will have their genome genetically and artificiallymanipulated so as to include genetic material which is not naturallypresent within the animal. For example, host animals include mice,hamsters and rats which have their endogenous PrP gene altered by theinsertion of an artificial gene of the present invention or by theinsertion of a native PrP gene of a genetically diverse test animal.

[0069] The terms “host animal” and “host mammal” are used to describeanimals which will have their genome genetically and artificiallymanipulated so as to include genetic material which is not naturallypresent within the animal. For example, host animals include mice,hamsters and rats which have their endogenous Dpl gene altered. In apreferred embodiment, these host animals also have a PrP gene altered bythe insertion of an artificial gene or by the insertion of a native PrPgene of a genetically diverse test animal.

[0070] The terms “test animal” and “test mammal” are used to describethe animal which is genetically diverse from the host animal in terms ofdifferences between the PrP gene of the host animal and the PrP gene ofthe test animal. The test animal may be any animal for which one wishesto run an assay test to determine whether a given sample contains prionswith which the test animal would generally be susceptible to infection.For example, the test animal may be a human, cow, sheep, pig, horse,cat, dog, turkey or chicken, and one may wish to determine whether aparticular sample includes prions which would normally only infect thetest animal. This is done by including PrP gene sequences of the testanimal into the host animal and inoculating the host animal with prionswhich would normally only infect the test animal.

[0071] The terms “genetically diverse animal” and “genetically diversemammal” are used to describe an animal which includes a native PrP codonsequence of the host animal which differs from the genetically diversetest animal by 17 or more codons, preferably 20 or more codons, and mostpreferably 28-40 codons. Thus, a mouse PrP gene is genetically diversewith respect to the PrP gene of a human, cow or sheep, but is notgenetically diverse with respect to the PrP gene of a hamster.

[0072] The terms “ablated PrP gene”, “disrupted PrP gene” and the likeare used interchangeably herein to mean an endogenous PrP gene which hasbeen altered (e.g., add and/or remove nucleotides) in a manner so as torender the gene inoperative. Examples of non-functional PrP genes andmethods of making such are disclosed in Büeler, et al, Nature356:577-582 (1992) which is incorporated herein by reference. Bothalleles of the genes are preferably disrupted.

[0073] The terms “ablated Dpl gene”, “disrupted Dpl gene” and the likeare used interchangeably herein to mean an endogenous Dpl gene which hasbeen altered (e.g., add and/or remove nucleotides) in a manner so as torender the gene inoperative.

[0074] The terms “hybrid animal”, “transgenic hybrid animal” and thelike are used interchangeably herein to mean an animal obtained from thecross-breeding of a first animal having altered Dpl activity and asecond animal which includes (1) an ablated endogenous PrP gene (1) achimeric or artificial PrP gene and/or (3) a PrP gene from a geneticallydiverse animal. For example a hybrid mouse is obtained by cross-breedinga mouse having a Dpl transgene knock-in with a mouse containing (1)bovine PrP genes (which may be present in high copy numbers) alone orwith (2) chimeric PrP genes. In another example, a hybrid mouse can beobtained by cross-breeding a PrP^(0/0) mouse with unregulated Dplactivity and a mouse having an inducible exogenous human PrP gene. Theterm hybrid includes any offspring of a hybrid including inbredoffspring of two hybrids provided the resulting offspring is susceptibleto infection with prions with normal infect only a genetically diversespecies and the symptoms of the infection are observable in about 350days or less, preferably 250 or less.

[0075] The term “incubation time” shall mean the time from inoculationof an animal with a prion until the time when the animal first developsdetectable symptoms of disease resulting from the infection. A reducedincubation time is preferably about 200 days “50 days or less, morepreferably about 50 days ” 20 days or less.

[0076] By “antibody” is meant an immunoglobulin protein which is capableof binding an antigen. Antibody as used herein is meant to include theentire antibody as well as any antibody fragments (e.g. F(ab′)₂, Fab′,Fab, Fv) capable of binding the epitope, antigen or antigenic fragmentof interest.

[0077] Antibodies of the invention are inmnunoreactive or immunospecificfor and therefore specifically and selectively bind to a Dpl protein.Antibodies for Dpl are preferably immunospecific—i.e., not substantiallycross-reactive with related materials. Although the term “antibody”encompasses all types of antibodies (e.g., monoclonal) the antibodies ofthe invention are preferably produced using the phage displaymethodology described herein. The preferred antibody of the invention isa purified antibody. By purified antibody is meant one which issufficiently free of other proteins, carbohydrates, and lipids withwhich it is naturally associated. Such an antibody “preferantiallybinds” to a Dpl protein (or an antigenic fragment thereof), i.e., doesnot substantially recognize and bind to other antigenically-unrelatedmolecules.

[0078] By “specifically activates”, as used herein, is meant an agentwhich activates Dpl or a fragment or analog thereof to initiateDpl-mediated biological events as described herein, but which does notsubstantially bind other molecules in a sample, e.g., a biologicalsample.

[0079] By “specifically inhibits”, as used herein, is meant an agentwhich inhibits activation of Dpl or a polypeptide, fragment or analogthereof. Preferably, the agent activates or inhibits the biologicalactivity in vivo or in vitro of the protein to which it binds.

[0080] By “substantial increase” is meant an increase in activity orother measurable phenotypic characteristic that is at leastapproximately a 2-fold increase over control level (where control assaysare performed in the absence of activator), preferably at leastapproximately a 5-fold increase, more preferably at least approximatelya 10-fold increase in activity over a control assay.

[0081] By “substantial decrease” or “substantial reduction” is meant adecrease or reduction in activity or other measurable phenotypiccharacteristic that is approximately 80% of the control level,preferably reduced to approximately 50% of the control level, or morepreferably reduced to approximately 10% or less of the control level.

[0082] The terms “screening method” and “assay method” are used todescribe a method of screening a candidate compound for its ability toact as an activator or suppressor of 1) Dpl activity and/or 2) prioninfection incubation time.

[0083] The terms “treatment”, “treating”, “treat” and the like are usedherein to generally mean obtaining a desired pharmacologic and/orphysiologic effect. The effect may be prophylactic in terms ofcompletely or partially preventing a disease or symptom thereof and/ormay be therapeutic in terms of a partial or complete cure for a diseaseand/or adverse effect attributable to the disease. “treatment” as usedherein covers any treatment of a disease in a mammal, particular ahuman, and includes:

[0084] (a) preventing the disease or symptom from occurring in a subjectwhich may be predisposed to the disease or symptom but has not yet beendiagnosed as having it;

[0085] (b) inhibiting the disease symptom, i.e., arresting itsdevelopment; or

[0086] (c) relieving the disease symptom, i.e., causing regression ofthe disease.

GENERAL ASPECTS OF THE INVENTION

[0087] Described herein is a novel 179 residue protein designated Dpl(Dpl) with ˜25% identity to all known prion proteins (PrP). Sincedatabase searches to identify PrP-like genes failed to be informative,and hybridization studies were not fruitful in such efforts (Westaway etal., Nucleic Acids Res. 14:2035-2044 (1986)), the sequencing of largecosmid clones containing the PrP gene was undertaken (Lee et al., GenomeRes. 8:1022-1037 (1998)). While most vertebrate genes of relatedfunction are not arranged in clusters, some related genes are. Instudies of the regions around the human, sheep and mouse PrP genes, noadditional open reading frames (ORF) were identified. Only when thesequencing of a cosmid clone isolated from a Prnp^(b/b) (ILN/J) mousewas extended downstream of PrP was a novel ORF found.

[0088] The Dpl locus, called “Prnd” is 16 Kb downstream of the PrP genePrnp and produces two major transcripts of 1.7 and 2.7 Kb as well assome unusual chimeric transcripts generated by intergenic splicing withPrnp. Polycistronic mRNA between PrP and Dpl have also been identified.Like PrP, Dpl mRNA is expressed during embryogenesis but in contrast toPrP, it is expressed at low levels in the CNS but at high levels in thetestis. Dpl is unregulated in the CNS of the two Prnp^(0/0) lines thatdevelop a late-onset ataxia and Purkinje cell degeneration, but not in aPrnp^(0/0) line that does not develop ataxia. The Dpl may be neurotoxicwhich explains why some lines of Prnp^(0/0) mice developneurodegeneration. Dpl may also be toxic to other non neuronal celltypes.

[0089] Moreover, the substantial homology between Dpl and PrP suggeststhese two proteins may share some biological properties and as such, Dplmay comprise a new element for understanding prion biology. Byidentifying Prnd as the first candidate for a PrP-like gene, we havebegun to define the Prn gene family. Interestingly, overexpression ofDpl, like some mutant and foreign PrPs, produces neurodegeneration inthe CNS of mice. In addition, Dpl and PrP may interact directly orindirectly by competing as ligands for a common receptor protein. Theinvention described herein provides a means for testing thesehypotheses.

[0090] Dpl Nucleic Acid

[0091] The terms “Dpl gene” and “Prnd” are used generically to designatethe coding region of Dpl. “Dpl gene” and “Prnd” are also intended tomean the open reading frame encoding specific Dpl polypeptides, introns,and adjacent 5′ and 3′ non-coding nucleotide sequences involved in theregulation of expression, up to about 1 kb beyond the transcribedregion, but possibly further in either direction. The DNA sequencesencoding Dpl may be cDNA or genomic DNA or a fragment thereof. The genemay be introduced into an appropriate vector for extrachromosomalmaintenance or for integration into the host.

[0092] The term “cDNA” as used herein is intended to include all nucleicacids that share the arrangement of sequence elements found in nativemature mRNA species, where sequence elements are exons (e.g., sequencesencoding open reading frames of the encoded polypeptide) and 3′ and 5′non-coding regions. Normally mRNA species have contiguous exons, withthe intervening introns removed by nuclear RNA splicing, to create acontinuous open reading frame encoding the Dpl polypeptide.

[0093] While other genomic Dpl sequences of other sources may havenon-contiguous open reading frames (e.g., where introns interrupt theprotein coding regions), the human genomic Dpl sequence has no intronsinterrupting the coding sequence. A genomic sequence of interestcomprises the nucleic acid present between the initiation codon and thestop codon, as defined in the listed sequences, including all of theintrons that are normally present in a native chromosome. It may furtherinclude the 3′ and 5′ untranslated regions found in the mature mRNA. Itmay further include specific transcriptional and translationalregulatory sequences, such as promoters, enhancers, etc., includingabout 1 kb, but possibly more, of flanking genomic DNA at either the 5′or 3′ end of the transcribed region. The genomic DNA may be isolated asa fragment of 100 kbp or smaller; and substantially free of flankingchromosomal sequence.

[0094] The sequence of this 5′ region, and further 5′ upstream sequencesand 3′ downstream sequences, may be utilized for promoter elements,including enhancer binding sites, that provide for expression in tissueswhere Dpl is expressed. The sequences of the Dpl promoter elements ofthe invention can be based on the nucleotide sequences of any species(e.g., mammalian or non-mammalian (e.g., reptiles, amphibians, avian(e.g., chicken)), particularly mammalian, including human, rodent (e.g.,murine or rat), bovine, ovine, porcine, murine, or equine, preferablyrat or human) and can be isolated or produced from any source whethernatural, synthetic, semi-synthetic or recombinant.

[0095] The tissue specific expression of Dpl is useful for determiningthe pattern of expression, and for providing promoters that mimic thenative pattern of expression. Naturally occurring polymorphisms in thepromoter region are useful for determining natural variations inexpression, particularly those that may be associated with disease.Alternatively, mutations may be introduced into the promoter region todetermine the effect of altering expression in experimentally definedsystems. Methods for the identification of specific DNA motifs involvedin the binding of transcriptional factors are known in the art, e.gsequence similarity to known binding motifs, gel retardation studies,etc. For examples, see Blackwell et al., Mol Med 1:194-205 (1995);Mortlock et al., Genome Res. 6: 327-33 (1996); and Joulin et al., Eur JBiochem 232: 620-626 (1995).

[0096] In one embodiment, the promoter is used to modulate Dplexpression. As discussed below, Dpl is expressed in embryonic cardiactissue and a subset of neurons in the adult brain. Thus, thedevelopmentally timed expression directed by the Dpl promoter can beexploited to facilitate expression of heterologous genes operably linkedto the Dpl promoter. Exemplary genes of interest that can be expressedfrom the Dpl promoter include, but are not necessarily limited to:marker genes (e.g., for marking the neuronal cells expressing Dpl) andreporter genes (e.g., luciferase, CAT, etc.) Such marker and reportergenes can be used to aid in elucidation of Dpl's normal physiologicalfunction, in identifying mechanisms for regulating Dpl and/or to searchfor bioactive agents (e.g., candidate pharmaceutical agents) thatregulate Dpl expression, and the like.

[0097] The regulatory sequences may be used to identify cis actingsequences required for transcriptional or translational regulation ofDpl expression, especially in different tissues or stages ofdevelopment, and to identify cis acting sequences and trans actingfactors that regulate or mediate Dpl expression. Such transcriptional ortranslational control regions may be operably linked to a Dpl gene orother genes in order to promote expression of wild type or altered Dplor other proteins of interest in cultured cells, or in embryonic, fetalor adult tissues, and for gene therapy. Dpl transcriptional ortranslational control regions can also be used to identify extracellularsignal molecules that regulate Dpl promoter activity, and thus regulateDpl expression.

[0098] The nucleic acid compositions used in the subject invention mayencode all or a part of the Dpl polypeptides as appropriate. The Dplsequences may be directed to forms of Dpl associated with disease statesand/or to naturally occurring variants of the protein. Fragments may beobtained of the DNA sequence by chemically synthesizing oligonucleotidesin accordance with conventional methods, by restriction enzymedigestion, by PCR amplification, etc. For the most part, DNA fragmentswill be of at least about ten contiguous nucleotides, usually at leastabout 15 nt, more usually at least about 18 nt to about 20 nt, moreusually at least about 25 nt to about 50 nt. Such small DNA fragmentsare useful as primers for PCR, hybridization screening, etc. Larger DNAfragments, i.e. greater than 100 nt are useful for production of theencoded polypeptide. For use in amplification reactions, such as PCR, apair of primers will be used. The exact composition of the primersequences is not critical to the invention, but for most applicationsthe primers will hybridize to the subject sequence under stringentconditions, as known in the art. It is preferable to choose a pair ofprimers that will generate an amplification product of at least about 50nt, preferably at least about 100 nt. Algorithms for the selection ofprimer sequences are generally known, and are available in commercialsoftware packages. Amplification primers hybridize to complementarystrands of DNA, and will prime towards each other.

[0099] The Dpl gene is isolated and obtained in substantial purity,generally as other than an intact mammalian chromosome. Usually, the DNAwill be obtained substantially free of other nucleic acid sequences thatdo not include a Dpl sequence or fragment thereof, generally being atleast about 50%, usually at least about 90% pure and are typically“recombinant”, i.e. flanked by one or more nucleotides with which it isnot normally associated on a naturally occurring chromosome.

[0100] The DNA sequences are used in a variety of ways. They may be usedas probes for identifying other genes encoding prion-like proteins, orfor identifying Dpl homologs in various species. Mammalian homologs havesubstantial sequence similarity to one another, i.e. at least 75%,usually at least 90%, more usually at least 95% sequence identity.Sequence similarity is calculated based on a reference sequence, whichmay be a subset of a larger sequence, such as a conserved motif, codingregion, flanking region, etc. A reference sequence will usually be atleast about 18 nt long, more usually at least about 30 nt long, and mayextend to the complete sequence that is being compared. Algorithms forsequence analysis are known in the art, such as BLAST, described inAltschul et al., J Mol Biol 215:403-10 (1990).

[0101] Nucleic acids having sequence similarity are detected byhybridization under low stringency conditions, for example, at 50 ° C.and 6 XSSC (0.9 M saline/0.09 M sodium citrate) and remain bound whensubjected to washing at 55 ° C. in 1 XSSC (0.15 M sodium chloride/0.015M sodium citrate). Sequence identity may be determined by hybridizationunder high stringency conditions, for example, at 50 ° C. or higher and0.1 XSSC (15 mM saline/0. 15 mM sodium citrate). By using probes,particularly labeled probes of DNA sequences, one can isolate homologousor related genes. The source of homologous genes may be any species,e.g. primate species, particularly human; rodents, such as rats andmice, canines, felines, bovines, ovines, equines, yeast, Drosophila,Caenhorabditis, etc.

[0102] The Dpl-encoding DNA may also be used to identify expression ofthe gene in a biological specimen. The manner in which one probes cellsfor the presence of particular nucleotide sequences, as genomic DNA orRNA, is well established in the literature and does not requireelaboration here. mRNA is isolated from a cell sample. mRNA may beamplified by RT-PCR, using reverse transcriptase to form a complementaryDNA strand, followed by polymerase chain reaction amplification usingprimers specific for the subject DNA sequences. Alternatively, mRNAsample is separated by gel electrophoresis, transferred to a suitablesupport, e.g. nitrocellulose, nylon, etc., and then probed with afragment of the subject DNA as a probe. Other techniques, such asoligonucleotide ligation assays, in situ hybridizations, andhybridization to DNA probes arrayed on a solid chip may also find use.Detection of mRNA hybridizing to a Dpl sequence is indicative of Dplgene expression in the sample.

[0103] The Dpl nucleic acid sequence may be modified for a number ofpurposes, particularly where they will be used intracellularly, forexample, by being joined to a nucleic acid cleaving agent, e.g. achelated metal ion, such as iron or chromium for cleavage of the gene,or the like.

[0104] The sequence of the Dpl locus, including flanking promoterregions and coding regions, may be mutated in various ways known in theart to generate targeted changes in promoter strength, sequence of theencoded protein, etc. The DNA sequence or product of such a mutationwill be substantially similar to the sequences provided herein, i.e.will differ by at least one nucleotide or amino acid, respectively, andmay differ by at least two but not more than about ten nucleotides oramino acids. The sequence changes may be substitutions, insertions ordeletions. Deletions may further include larger changes, such asdeletions of a domain or exon. Other modifications of interest includeepitope tagging, e.g. with the FLAG system, HA, etc. For studies ofsubcellular localization, fusion proteins with green fluorescentproteins (GFP) may be used. Such mutated genes may be used to studystructure-function relationships of Dpl polypeptides with otherpolypeptides (e.g., Nkx-6.1, which is co-expressed with Dpl), or toalter properties of the proteins that affect their function orregulation. Such modified Dpl sequences can be used to, for example,generate the transgenic animals.

[0105] Techniques for in vitro mutagenesis of cloned genes are known.Examples of protocols for scanning mutations may be found in Gustin etal., Biotechniques 14:22 (1993); Barany, Gene 37:111-23 (1985);Colicelli et al., Mol Gen Genet 199:537-9 (1985); and Prentki et al.,Gene 29:303-13 (1984). Methods for site specific mutagenesis can befound in Sambrook et al., Molecular Cloning: A Laboratory Manual, CSHPress, pp. 15.3-15.108 (1989); Weiner et al., Gene 126:35-41 (1993);Sayers et al., Biotechniques 13:592-6 (1992); Jones et al.,Biotechniques 12:528-30 (1992); Barton et al., Nucleic Acids Res18:7349-55 (1990); Barotti et al., Gene Anal Tech 6:67-70 (1989); andZhu, Anal Biochem 177:120-4 (1989).

[0106] Dpl gene comprising portions of the host animal e.g., endportions of a host animal and a middle portion of a genetically diversetest animal wherein the middle portion includes a specific alterationsdesigned to match that of a disease state of such a host. Further, theinvention includes, a transgenic animal containing the artificial geneor modulated expression of a Dpl gene from a genetically diverse animal,hybrid transgenic animals which are the offspring of differenttransgenic animals with each other or with a transgenic animal that hasan ablated endogenous prion protein gene, a standardized prionpreparation and assay methodology which uses the preparation andgenetically altered animals to detect pathogenic prions in a sample.

[0107] The artificial gene includes a sequence such that when it isinserted into the genome of an animal (such as a mouse), the animal isrendered susceptible to infection with prions which normally wouldinfect only a specific species of genetically diverse animal (such as ahuman, cow, sheep, pig, chicken, cat or dog). The artificial Dpl genemay be comprised partially or completely of an artificial polynucleotidesequence, i.e. codon sequences not present in any native Dpl genesequence. Alternatively, the artificial gene may be comprised of thecodon sequence of a host animal with one or more codon substitutionsbeing made wherein the substitutions are preferably corresponding Dplgene codons from a genetically diverse animal, meaning that Dpl genediffers from the Dpl gene of the host animal by 20 or more codons.Transgenic animals containing elevated levels of expression of the Dplgene which can be obtained for example, by over expression of the genewith an enhanced promoter and/or with high copy numbers of the naturalDpl gene of a genetically diverse animal are also disclosed. Hybridtransgenic animals include animals resulting from a cross between twotransgenic animals and in particular a cross between a transgenic animalcontaining the entire prion protein gene of a genetically diverse animal(e.g., a mouse containing a human prion protein gene) and an animal withits endogenous prion protein gene disrupted (e.g., a mouse with anablated prion protein gene). Hybrids also specifically include crossinga transgenic animal having a chimeric prion protein gene with an animalwith its endogenous prion protein gene ablated.

[0108] Dpl Transgenic Animals

[0109] The Dpl-encoding nucleic acids can be used to generategenetically modified non-human animals or site specific genemodifications in cell lines. The term “transgenic” is intended toencompass genetically modified animals having a deletion or otherknock-out of Dpl gene activity, having an exogenous Dpl gene that isstably transmitted in the host cells, “knock-in” having altered Dpl geneexpression, or having an exogenous Dpl promoter operably linked to areporter gene. Of particular interest are homozygous and heterozygousknock-outs of Dpl.

[0110] Transgenic animals may be made through homologous recombination,where the Dpl locus is altered. Alternatively, a nucleic acid constructis randomly integrated into the genome. Vectors for stable integrationinclude plasmids, retroviruses and other animal viruses, YACs, and thelike. Of interest are transgenic mammals, preferably a mammal from agenus selected from the group consisting of Mus (e.g., mice), Rattus(e.g., rats), Oryctologus (e.g., rabbits) and Mesocricetus (e.g.,hamsters). More preferably the animal is a mouse which is defective orcontains some other alteration in Dpl gene expression or function.

[0111] A “knock-out” animal is genetically manipulated to substantiallyreduce, or eliminate endogenous Dpl function, preferably such thattarget gene expression is undetectable or insignificant. Differentapproaches may be used to achieve the “knock-out”. A chromosomaldeletion of all or part of the native Dpl homolog may be induced.Deletions of the non-coding regions, particularly the promoter region,3′ regulatory sequences, enhancers, or deletions of gene that activateexpression of the Dpl genes. A functional knock-out may also be achievedby the introduction of an anti-sense construct that blocks expression ofthe native Dpl gene (for example, see Li et al., Cell 85:319-329(1996)).

[0112] Conditional knock-outs of Dpl gene function can also begenerated. Conditional knock-outs are transgenic animals that exhibit adefect in Dpl gene function upon exposure of the animal to a substancethat promotes target gene alteration, introduction of an enzyme thatpromotes recombination at the target gene site (e.g., Cre in theCre-loxP system), or other method for directing the target genealteration.

[0113] For example, a transgenic animal having a conditional knock-outof Dpl gene function can be produced using the Cre-loxP recombinationsystem (see, e.g., Kilby et al., Trends Genet 9:413-421 (1993)). Cre isan enzyme that excises the DNA between two recognition sequences, termedloxP. This system can be used in a variety of ways to create conditionalknock-outs of Dpl. For example, two independent transgenic mice can beproduced: one transgenic for a Dpl sequence flanked by loxP sites and asecond transgenic for Cre. The Cre transgene can be under the control ofan inducible or developmentally regulated promoter (Gu et al., Cell73:1155-1164 (1993); Gu et al., Science 265:103-106 (1994)), or undercontrol of a tissue-specific or cell type-specific promoter (e.g., aneuron-specific promoter or brain tissue-specific promoter). The Dpltransgenic is then crossed with the Cre transgenic to produce progenydeficient for the Dpl gene only in those cells that expressed Cre duringdevelopment.

[0114] Transgenic animals may be made having an exogenous Dpl gene. Forexample, the transgenic animal may comprise a “knock-in” of a Dpl gene,such that the host cell genome contains an alteration that results inaltered expression (e.g., increased (including ectopic) or decreasedexpression) of a Dpl gene, e.g., by introduction of an additional copyof the target gene, or by operatively inserting a regulatory sequencethat provides for enhanced expression of an endogenous copy of thetarget gene. “knock-in” transgenics can be transgenic animals having aheterozygous knock-in of the Dpl gene or a homozygous knock-in of theDpl. “Knock-ins” also encompass conditional knock-ins.

[0115] The exogenous gene introduced into the host cell genome toproduce a transgenic animal is usually either from a different speciesthan the animal host, or is otherwise altered in its coding ornon-coding sequence. The introduced gene may be a wild-type gene,naturally occurring polymorphism, or a genetically manipulated sequence,for example those previously described with deletions, substitutions orinsertions in the coding or non-coding regions. The introduced sequencemay encode a Dpl polypeptide, or may utilize the Dpl promoter operablylinked to a reporter gene. Where the introduced gene is a codingsequence, it is usually operably linked to a promoter, which may beconstitutive or inducible, and other regulatory sequences required forexpression in the host animal.

[0116] Specific constructs of interest include, but are not limited to,anti-sense Dpl, or a ribozyme based on a Dpl sequence, which will blockDpl expression, as well as expression of dominant negative Dplmutations, and over-expression of a Dpl gene. A detectable marker, suchas lac Z may be introduced into the Dpl locus, where upregulation ofexpression of the corresponding Ngn gene will result in an easilydetected change in phenotype. Constructs utilizing a promoter region ofthe Dpl genes in combination with a reporter gene or with the codingregion of Dpl are also of interest. Constructs having a sequenceencoding a truncated or altered (e.g, mutated) Dpl are also of interest.

[0117] The modified cells or animals are useful in the study of functionand regulation of Dpl and other proteins involved the CNS and embryonicdevelopment. Such modified cells or animals are also useful in, forexample, the study of the function and regulation of neurotoxic and/orneurodegenerative processes affected by Dpl, as well as the study of theprion family of genes in vivo. Thus, the transgenic animals of theinvention are useful in identifying both downstream targets of Dpl andpotentially a receptor for Dpl, as such may have a role in thephenotypes associated with defects in Dpl.

[0118] Animals may also be used in functional studies, drug screening,etc., e.g. to determine the effect of a candidate drug on embryonicdevelopment, neurodegeneration, or on symptoms associated with diseaseor conditions associated with Dpl defects (e.g., on symptoms associatedwith neurotoxicity. A series of small deletions and/or substitutions maybe made in the Dpl genes to determine the role of differentpolypeptide-encoding regions in DNA binding, transcriptional regulation,etc. By providing expression of Dpl protein in cells in which it isotherwise not normally produced (e.g., ectopic expression), one caninduce changes in cell behavior. These animals are also useful forexploring mechanistic models of neurodegeneration, and in particular themechanisms involved in plaque formation, e.g. PrP^(Sc) plaques in thebrain.

[0119] DNA constructs for homologous recombination will comprise atleast a portion of the Dpl gene with the desired genetic modification,and will include regions of homology to the target locus. DNA constructsfor random integration need not include regions of homology to mediaterecombination. Conveniently, markers for positive and negative selectionare included. Methods for generating cells having targeted genemodifications through homologous recombination are known in the art. Forvarious techniques for transfecting mammalian cells, see Keown et al.,Methods in Enzymology 185:527-537 (1990).

[0120] For embryonic stem (ES) cells, an ES cell line may be employed,or embryonic cells may be obtained freshly from a host, e.g. mouse, rat,guinea pig, etc. Such cells are grown on an appropriatefibroblast-feeder layer or grown in the presence of appropriate growthfactors, such as leukemia inhibiting factor (LIF). When ES cells havebeen transformed, they may be used to produce transgenic animals. Aftertransformation, the cells are plated onto a feeder layer in anappropriate medium. Cells containing the construct may be detected byemploying a selective medium. After sufficient time for colonies togrow, they are picked and analyzed for the occurrence of homologousrecombination or integration of the construct. Those colonies that arepositive may then be used for embryo manipulation and blastocystinjection. Blastocysts are obtained from 4 to 6 week old superovulatedfemales. The ES cells are trypsinized, and the modified cells areinjected into the blastocoel of the blastocyst. After injection, theblastocysts are returned to each uterine horn of pseudopregnant females.Females are then allowed to go to term and the resulting littersscreened for mutant cells having the construct. By providing for adifferent phenotype of the blastocyst and the ES cells, chimeric progenycan be readily detected.

[0121] The chimeric animals are screened for the presence of themodified gene. Chimeric animals having the modification (normallychimeric males) are mated with wildtype animals to produceheterozygotes, and the heterozygotes mated to produce homozygotes, Ifthe gene alterations cause lethality at some point in development,tissues or organs can be maintained as allogeneic or congenic grafts ortransplants, or in in vitro culture.

[0122] Production of an overt morphological defect in single or doublegene ablated mice may pave the way for precise assignment of Dpl andPrP^(c) functions. The role of Dpl overexpression in Purkinje celldegeneration or alternatively, of closely linked genes present withinextant Prn BAC and YAC clones (Westaway et al., Proc. Natl. Acad. Sci.USA 91, 6418-6422 (1994)) can be assessed by use of heterologouspromoter elements. It will be of interest to test the susceptibility ofmice, which overexpress Dpl or which harbor null or mutant alleles ofPrnD to infection with prions.

[0123] Investigation of genetic function may also utilize non-mammalianmodels, particularly using those organisms that are biologically andgenetically well-characterized, such as C. elegans, D. melanogaster andS. cerevisiae. For example, transposon (Tc1) insertions in the nematodehomolog of a Dpl gene or a promoter region of a Dpl gene may be made.The Dpl gene sequences may be used to knock-out or to complement definedgenetic lesions in order to determine the physiological and biochemicalpathways involved in function of prion-like proteins. It is well knownthat human genes can complement mutations in lower eukaryotic models.

[0124] Production of Dpl Polypeptides

[0125] Dpl-encoding nucleic acid may be employed to synthesizefull-length Dpl polypeptides or fragments thereof, particularlyfragments corresponding to functional domains; DNA binding sites; etc.;and including fusions of the subject polypeptides to other proteins orparts thereof. For expression, an expression cassette may be employed,providing for a transcriptional and translational initiation region,which may be inducible or constitutive, where the coding region isoperably linked under the transcriptional control of the transcriptionalinitiation region, and a transcriptional and translational terminationregion. Various transcriptional initiation regions may be employed thatare functional in the expression host.

[0126] The polypeptides may be expressed in prokaryotes or eukaryotes inaccordance with conventional ways, depending upon the purpose forexpression. For large scale production of the protein, a unicellularorganism, such as E. coli, B. subtilis, S. cerevisiae, or cells of ahigher organism such as vertebrates, particularly mammals, e.g. COS 7cells, may be used as the expression host cells. In many situations, itmay be desirable to express the Dpl genes in mammalian cells, especiallywhere the encoded polypeptides will benefit from native folding andpost-translational modifications. Small peptides can also be synthesizedin the laboratory.

[0127] With the availability of the polypeptides in large amounts, byemploying an expression host, the polypeptides may be isolated andpurified in accordance with conventional ways. A lysate may be preparedof the expression host and the lysate purified using HPLC, exclusionchromatography, gel electrophoresis, affinity chromatography, or otherpurification technique. The purified polypeptide will generally be atleast about 80% pure, preferably at least about 90% pure, and may be upto and including 100% pure. Pure is intended to mean free of otherproteins, as well as cellular debris.

[0128] The Dpl polypeptides can be used for the production ofantibodies, where short fragments provide for antibodies specific forthe particular polypeptide, and larger fragments or the entire proteinallow for the production of antibodies over the surface of thepolypeptide. Antibodies may be raised to the wild-type or variant formsof Dpl, including forms of Dpl that may be associated with diseasestates or naturally occurring allelic variants. Antibodies may be raisedto isolated peptides corresponding to these domains, or to the nativeprotein, e.g by immunization with cells expressing Dpl, immunizationwith liposomes having Dpl polypeptides inserted in the membrane, etc.

[0129] Antibodies are prepared in accordance with conventional ways,where the expressed polypeptide or protein is used as an immunogen, byitself or conjugated to known immunogenic carriers, e.g. KLH, pre-SHBsAg, other viral or eukaryotic proteins, or the like. Variousadjuvants may be employed, with a series of injections, as appropriate.For monoclonal antibodies, after one or more booster injections, thespleen is isolated, the lymphocytes immortalized by cell fusion, andthen screened for high affinity antibody binding. The immortalizedcells, i.e. hybridomas, producing the desired antibodies may then beexpanded. For further description, see Monoclonal Antibodies: ALaboratory Manual, Harlow and Lane eds., Cold Spring HarborLaboratories, Cold Spring Harbor, N.Y., 1988. If desired, the mRNAencoding the heavy and light chains may be isolated and mutagenized bycloning in E. coli, and the heavy and light chains mixed to furtherenhance the affinity of the antibody. Alternatives to in vivoimmunization as a method of raising antibodies include binding to phage“display” libraries, usually in conjunction with in vitro affinitymaturation.

[0130] Isolation of Dpl Allelic Variants and Homologues in Other Species

[0131] Other mammalian Dpl genes can be identified and their functioncharacterized using the Dpl genes used in the present invention. Dplgenes of interest include, but are not limited to, mammalian (e.g.,human, rodent (e.g, murine, or rat), bovine, feline, canine, and thelike) and non-mammalian (e.g., chicken, reptile, and the like). Methodsfor identifying, isolating, sequencing, and characterizing an unknowngene based upon its homology to a known gene sequence are well known inthe art (see, e.g., Sambrook et al., Molecular Cloning: A LaboratoryManual, CSH Press 1989).

[0132] Drug Screening

[0133] The animal models of the invention, as well as methods using theDpl polypeptides in vitro, can be used to identify candidate agents thataffect Dpl expression (e.g., by affecting Dpl promoter function) or thatinteract with Dpl polypeptides. Agents of interest can include thosethat enhance, inhibit, regulate, or otherwise affect Dpl activity and/orexpression. Agents that alter Dpl activity and/or expression can beused, for example: to treat or study disorders associated with increasedDpl activity (e.g., neurodegenerative disorders); to treat variousdegenerative or developmental conditions, e.g. inappropriate neuraloutgrowth or other conditions that are altered by a change in Dplexpression, activity, and/or conformation; and to facilitate developmentof PrP^(Sc) either in vitro or in vivo. Candidate agents includesynthetic molecules (e.g., small molecule drugs, peptides, or othersynthetically produced molecules or compounds, as well as recombinantlyproduced gene products) as well as naturally-occurring compounds (e.g.,polypeptides, endogenous factors, plant extracts, and the like).

[0134] Drug Screening Assays

[0135] Of particular interest in the present invention is theidentification of agents that have activity in affecting Dpl expressionand/or function. Such agents are candidates for development oftreatments for, for example, increased and/or inappropriate neuraloutgrowth, or other condition that may be associated with altered Dplactivity. Drug screening identifies agents that provide a replacement orenhancement for Dpl function in affected cells. Conversely, agents thatreverse or inhibit Dpl function may provide a means to regulateneurodegeneration or neurotoxicity, especially in Purkinje cells. Ofparticular interest are screening assays for agents that have a lowtoxicity for human cells.

[0136] The term “agent” as used herein describes any molecule, e.g.protein or pharmaceutical, with the capability of altering or mimickingthe expression or physiological function of Dpl. Generally a pluralityof assay mixtures are run in parallel with different agentconcentrations to obtain a differential response to the variousconcentrations. Typically, one of these concentrations serves as anegative control, i.e. at zero concentration or below the level ofdetection.

[0137] Candidate agents encompass numerous chemical classes, thoughtypically they are organic molecules, preferably small organic compoundshaving a molecular weight of more than 50 and less than about 2,500daltons. Candidate agents comprise functional groups necessary forstructural interaction with proteins, particularly hydrogen bonding, andtypically include at least an amine, carbonyl, hydroxyl or carboxylgroup, preferably at least two of the functional chemical groups. Thecandidate agents often comprise cyclical carbon or heterocyclicstructures and/or aromatic or polyaromatic structures substituted withone or more of the above functional groups. Candidate agents are alsofound among biomolecules including, but not limited to: peptides,saccharides, fatty acids, steroids, purines, pyrimidines, derivatives,structural analogs or combinations thereof.

[0138] Candidate agents are obtained from a wide variety of sourcesincluding libraries of synthetic or natural compounds. For example,numerous means are available for random and directed synthesis of a widevariety of organic compounds and biomolecules, including expression ofrandomized oligonucleotides and oligopeptides. Alternatively, librariesof natural compounds in the form of bacterial, fungal, plant and animalextracts are available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs.

[0139] Screening of Candidate Agents In vivo

[0140] Agents can be screened for their ability to affect Dpl expressionor function or to mitigate an undesirable phenotype (e.g., a symptom)associated with an alteration in Dpl expression or function. In apreferred embodiment, screening of candidate agents is performed in vivoin a transgenic animal described herein. Transgenic animals suitable foruse in screening assays include any transgenic animal having analteration in Dpl expression, and can include transgenic animals having,for example, an exogenous and stably transmitted human Dpl genesequence, a reporter gene composed of a (removed human) Dpl promotersequence operably linked to a reporter gene (e.g., β-galactosidase, CAT,or other gene that can be easily assayed for expression), or ahomozygous or heterozygous knockout of a Dpl gene. The transgenicanimals can be either homozygous or heterozygous for the geneticalteration and, where a sequence is introduced into the animal's genomefor expression, may contain multiple copies of the introduced sequence.Where the in vivo screening assay is to identify agents that affect theactivity of the Dpl promoter, the Dpl promoter can be operably linked toa reporter gene (e.g., luciferase) and integrated into the non-humanhost animal's genome or integrated into the genome of a culturedmammalian cell line.

[0141] The candidate agent is administered to a non-human, transgenicanimal having altered Dpl expression, and the effects of the candidateagent determined. The candidate agent can be administered in any mannerdesired and/or appropriate for delivery of the agent in order to effecta desired result. For example, the candidate agent can be administeredby injection (e.g., by injection intravenously, intramuscularly,subcutaneously, or directly into the tissue in which the desired affectis to be achieved), orally, or by any other desirable means. Normally,the in vivo screen will involve a number of animals receiving varyingamounts and concentrations of the candidate agent (from no agent to anamount of agent hat approaches an upper limit of the amount that can bedelivered successfully to the animal), and may include delivery of theagent in different formulation. The agents can be administered singly orcan be combined in combinations of two or more, especially whereadministration of a combination of agents may result in a synergisticeffect.

[0142] The effect of agent administration upon the transgenic animal canbe monitored by assessing Dpl function as appropriate (e.g., byexamining expression of a reporter or fusion gene), or by assessing aphenotype associated with the Dpl expression. For example, where thetransgenic animal used in the screen contains a defect in Dpl expression(e.g., due to a knock-out of the gene), the effect of the candidateagent can be assessed by determining levels of neurodegeneration in thecontrol mouse relative to the levels produced in the Dpl defectivetransgenic mouse and/or in wildtype mice (e.g, by assessing levels ofataxia in the treated mice). Methods for assaying neurological defectsin rodent are well known in the art. Where the in vivo screening assayis to identify agents that affect the activity of the Dpl promoter andthe non-human transgenic animal (or cultured mammalian cell line)comprises a Dpl promoter operably linked to a reporter gene, the effectsof candidate agents upon Dpl promoter activity can be screened by, forexample, monitoring the expression from the Dpl promoter (throughdetection of the reporter gene) and correlation of altered Dpl promoteractivity with neuronal activity and/or degeneration. Alternatively or inaddition, Dpl promoter activity can be assessed by detection(qualitative or quantitative) of Dpl mRNA or protein levels. Where thecandidate agent affects Dpl expression, and/or affects a Dpl-associatedphenotype, in a desired manner, the candidate agent is identified as anagent suitable for use in therapy of a Dpl-associated disorder or aPrP-associated disorder.

[0143] Screening of Candidate Agents In vitro

[0144] In addition to screening of agents in Dpl transgenic animals, awide variety of in vitro assays may be used for this purpose, includinglabeled in vitro protein-protein binding assays, protein-DNA bindingassays, electrophoretic mobility shift assays, immunoassays for proteinbinding, and the like. For example, by providing for the production oflarge amounts of Dpl protein, one can identify ligands or substratesthat bind to, modulate or mimic the action of the proteins. The purifiedprotein may also be used for determination of three-dimensional crystalstructure, which can be used for modeling intermolecular interactions,transcriptional regulation, etc.

[0145] The screening assay can be a binding assay, wherein one or moreof the molecules may be joined to a label, and the label directly orindirectly provide a detectable signal. Various labels includeradioisotopes, fluorescers, chemiluminescers, enzymes, specific bindingmolecules, particles, e.g. magnetic particles, and the like. Specificbinding molecules include pairs, such as biotin and streptavidin,digoxin and antidigoxin etc. For the specific binding members, thecomplementary member would normally be labeled with a molecule thatprovides for detection, in accordance with known procedures.

[0146] A variety of other reagents may be included in the screeningassays described herein. Where the assay is a binding assay, theseinclude reagents like salts, neutral proteins, e.g. albumin, detergents,etc that are used to facilitate optimal protein-protein binding,protein-DNA binding, and/or reduce non-specific or backgroundinteractions. Reagents that improve the efficiency of the assay, such asprotease inhibitors, nuclease inhibitors, anti-microbial agents, etc.may be used. The mixture of components are added in any order thatprovides for the requisite binding. Incubations are performed at anysuitable temperature, typically between 4 and 40 ° C. Incubation periodsare selected for optimum activity, but may also be optimized tofacilitate rapid high-throughput screening. Typically between 0.1 and 1hours will be sufficient.

[0147] Other assays of interest detect agents that mimic Dpl function.For example, candidate agents are added to a cell that lacks functionalDpl, and screened for the ability to reproduce Dpl activity in afunctional assay. In a particular embodiment, agents that mimicunregulated Dpl activity are screened for the ability to decreaseincubation time of prion disorders.

[0148] Many mammalian genes have homologs in yeast and lower animals.The study of such homologs' physiological role and interactions withother proteins in vivo or in vitro can facilitate understanding ofbiological function. In addition to model systems based on geneticcomplementation, yeast has been shown to be a powerful tool for studyingprotein-protein interactions through the two hybrid system described inChien et al., Proc. Natl. Acad. Sci. USA 88:9578-9582 (1991). Two-hybridsystem analysis is of particular interest for exploring transcriptionalactivation by Dpl proteins and to identify cDNAs encoding polypeptidesthat interact with Dpl. Binding to Dpl may influence thefunction/activity of particular target proteins or be itselffunctionally modulated and may bear relevance for both normal orspecific disease states.

[0149] Identified Candidate Agents

[0150] The compounds having the desired pharmacological activity may beadministered in a physiologically acceptable carrier to a host fortreatment of a condition attributable or in part modulated by a defectin normal Dpl function (e.g., a neurological disorder associated withunregulated Dpl levels. The compounds may also be used to enhance Dplfunction. The therapeutic agents may be administered in a variety ofways, orally, topically, parenterally e.g. subcutaneously,intraperitoneally, by viral infection, intravascularly, etc. Inhaledtreatments are of particular interest. Depending upon the manner ofintroduction, the compounds may be formulated in a variety of ways. Theconcentration of therapeutically active compound in the formulation mayvary from about 0.1-100 wt. %.

[0151] The pharmaceutical compositions can be prepared in various forms,such as granules, tablets, pills, suppositories, capsules, suspensions,salves, lotions and the like. Pharmaceutical grade organic or inorganiccarriers and/or diluents suitable for oral and topical use can be usedto make up compositions containing the therapeutically-active compounds.Diluents known to the art include aqueous media, vegetable and animaloils and fats. Stabilizing Agents, wetting and emulsifying Agents, saltsfor varying the osmotic pressure or buffers for securing an adequate pHvalue, and skin penetration enhancers can be used as auxiliary agents.

[0152] Pharmacogenetics

[0153] Pharmacogenetics is the linkage between an individual's genotypeand that individual's ability to metabolize or react to a therapeuticagent. Differences in metabolism or target sensitivity can lead tosevere toxicity or therapeutic failure by altering the relation betweenbioactive dose and blood concentration of the drug. In the past fewyears, numerous studies have established good relationships betweenpolymorphisms in metabolic enzymes or drug targets, and both responseand toxicity. These relationships can be used to individualizetherapeutic dose administration.

[0154] Genotyping of polymorphic alleles is used to evaluate whether anindividual will respond well to a particular therapeutic regimen. Thepolymorphic sequences are also used in drug screening assays, todetermine the dose and specificity of a candidate therapeutic agent. Acandidate Dpl polymorphism is screened with a target therapy todetermine whether there is an influence on the effectiveness intreating, for example, a neurological disorder. Drug screening assaysare performed as described above. Typically two or more differentsequence polymorphisms are tested for response to a therapy.

[0155] Where a particular sequence polymorphism correlates withdifferential drug effectiveness, diagnostic screening may be performed.Diagnostic methods have been described in detail in a preceding section.The presence of a particular polymorphism is detected, and used todevelop an effective therapeutic strategy for the affected individual.

[0156] Detection of Dpl Associated Disorders

[0157] Detection of Dpl levels and/or the presence of differentconformations of Dpl can provide a means for diagnosingneurodegenerative disorders associated with Dpl. In particular,detection of changes in Dpl expression and/or changes in Dplconformation may be associated with disorders involving Purkinje celldegeneration, such as hereditary cerebellar cortical atrophy,generalized epilepsy with tonic-clonic seizures (GTCS), spinocerebellarataxia type 6, and the like. Measurement of Dpl levels and/or differentconformations of the protein may also be used for otherneurodegenerative disorders, as will be apparent to one skilled in theart upon reading the present disclosure.

[0158] Diagnosis of Dpl-associated disorders is performed by protein,DNA or RNA sequence and/or hybridization analysis of any convenientsample from a patient, e.g. biopsy material, blood sample, scrapingsfrom cheek, etc. A nucleic acid sample from a patient having a disorderthat may be associated with Dpl is analyzed for the presence of apredisposing polymorphism in Dpl. A typical patient genotype will haveat least one predisposing mutation on at least one chromosome. Thepresence of a polymorphic Dpl sequence that affects the activity orexpression of the gene product, and confers an increased susceptibilityto a Dpl associated disorder (e.g, a neurodegenerative disorder) isconsidered a predisposing polymorphism. Individuals are screened byanalyzing their DNA or mRNA for the presence of a predisposingpolymorphism, as compared to sequence from an unaffected individual(s).

[0159] Screening may also be based on the functional or antigeniccharacteristics of the protein. Immunoassays designed to detectpredisposing polymorphisms in Dpl proteins may be used in screening.Where many diverse mutations lead to a particular disease phenotype,functional protein assays can be effective screening tools.

[0160] Biochemical studies may be performed to determine whether acandidate sequence polymorphism in the Dpl coding region or controlregions is associated with disease. For example, a change in thepromoter or enhancer sequence that affects expression of Dpl may resultin predisposition to neurological disorders, e.g. prion-mediateddisorders. Expression levels of a candidate variant allele are comparedto expression levels of the normal allele by various methods known inthe art. Methods for determining promoter or enhancer strength includequantitation of the expressed natural protein; insertion of the variantcontrol element into a vector with a reporter gene such asβ-galactosidase, luciferase, chloramphenicol acetyltransferase, etc.that provides for convenient quantitation; and the like. The activity ofthe encoded Dpl protein may be determined by comparison with thewild-type protein.

[0161] A number of methods are available for analyzing nucleic acids forthe presence of a specific sequence. Where large amounts of DNA areavailable, genomic DNA is used directly. Alternatively, the region ofinterest is cloned into a suitable vector and grown in sufficientquantity for analysis. Cells that express Dpl genes may be used as asource of mRNA, which may be assayed directly or reverse transcribedinto cDNA for analysis. The nucleic acid may be amplified byconventional techniques, such as the polymerase chain reaction (PCR), toprovide sufficient amounts for analysis. The use of the polymerase chainreaction is described in Saiki, et al., Science 239:487 (1985); a reviewof current techniques may be found in Sambrook, et al., MolecularCloning: A Laboratory Manual, CSH Press 1989, pp.14.2B 14.33.Amplification may also be used to determine whether a polymorphism ispresent, by using a primer that is specific for the polymorphism.Alternatively, various methods are known in the art that utilizeoligonucleotide ligation as a means of detecting polymorphisms, forexamples see Riley et al., Nucl. Acid Res. 18:2887-2890 (1990); andDelahunty et al., Am. J. Hum. Genet. 58:1239-1246 (1996).

[0162] A detectable label may be included in an amplification reaction.Suitable labels include fluorochromes, e.g. fluorescein isothiocyanate(FITC), rhodamine, Texas Red, phycoerythrin, allophycocyanin,6-carboxyfluorescein (6-FAM), 2′,7′-dimethoxy-4′,5′-dichloro-6-carboxyfluorescein (JOE),6-carboxy-X-rhodamine (ROX), 6-carboxy-2′, 4′, 7′, 4,7-hexachlorofluorescein (HEX), 5-carboxyfluorescein (5-FAM) or N, N, N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), radioactive labels, e.g. ³²P,³³P, ³⁵S, ³H; etc. The label may be a two stage system, where theamplified DNA is conjugated to biotin, haptens, etc. having a highaffinity binding partner, e.g. avidin, specific antibodies, etc., wherethe binding partner is conjugated to a detectable label. The label maybe conjugated to one or both of the primers. Alternatively, the pool ofnucleotides used in the amplification is labeled, so as to incorporatethe label into the amplification product.

[0163] The sample nucleic acid, e.g. amplified or cloned fragment, isanalyzed by one of a number of methods known in the art. The nucleicacid may be sequenced by dideoxy or other methods, and the sequence ofbases compared to either a neutral Dpl sequence (e.g., a Dpl sequencefrom an unaffected individual). Hybridization with the variant sequencemay also be used to determine its presence, by Southern blots, dotblots, etc. The hybridization pattern of a control and variant sequenceto an array of oligonucleotide probes immobilized on a solid support, asdescribed in U.S. Pat. No. 5,445,934, or in WO 95/35505, may also beused as a means of detecting the presence of variant sequences. Singlestrand conformational polymorphism (SSCP) analysis, denaturing gradientgel electrophoresis (DGGE), mismatch cleavage detection, andheteroduplex analysis in gel matrices are used to detect conformationalchanges created by DNA sequence variation as alterations inelectrophoretic mobility. Alternatively, where a polymorphism creates ordestroys a recognition site for a restriction endonuclease (restrictionfragment length polymorphism, RFLP), the sample is digested with thatendonuclease, and the products size fractionated to determine whetherthe fragment was digested. Fractionation is performed by gel orcapillary electrophoresis, particularly acrylamide or agarose gels.

[0164] The hybridization pattern of a control and variant sequence to anarray of oligonucleotide probes immobilized on a solid support, asdescribed in U.S. Pat. No. 5,445,934, or in WO 95/35505, may be used asa means of detecting the presence of variant sequences. In oneembodiment of the invention, an array of oligonucleotides are provided,where discrete positions on the array are complementary to at least aportion of mRNA or genomic DNA of the Dpl locus. Such an array maycomprise a series of oligonucleotides, each of which can specificallyhybridize to a nucleic acid sequence, e.g,. mRNA, cDNA, genomic DNA,etc. from the Dpl locus. Usually such an array will include at least 2different polymorphic sequences, i.e. polymorphisms located at uniquepositions within the locus, usually at least about 5, more usually atleast about 10, and may include as many as 50 to 100 differentpolymorphisms. The oligonucleotide sequence on the array will usually beat least about 12 nt in length, may be the length of the providedpolymorphic sequences, or may extend into the flanking regions togenerate fragments of 100 to 200 nt in length. For examples of arrays,see Hacia et al., Nature Genetics 14:441-447 (1996); Lockhart et al.,Nature Biotechnol. 14:1675-1680 (1996); and De Risi et al., NatureGenetics 14:457-460 (1996).

[0165] Antibodies specific for Dpl polymorphisms may be used inscreening immunoassays. A reduction or increase in Dpl and/or presenceof a Dpl disorder associated polymorphism is indicative that thesuspected disorder is Dpl-associated. A sample is taken from a patientsuspected of having a Dpl-associated disorder. Samples, as used herein,include tissue biopsies, biological fluids, organ or tissue culturederived fluids, and fluids extracted from physiological tissues, as wellas derivatives and fractions of such fluids. The number of cells in asample will generally be at least about 10³, usually at least 10⁴ moreusually at least about 10⁵. The cells may be dissociated, in the case ofsolid tissues, or tissue sections may be analyzed. Alternatively alysate of the cells may be prepared.

[0166] Diagnosis may be performed by a number of methods. The differentmethods all determine the absence or presence or altered amounts ofnormal or abnormal Dpl in patient cells suspected of having apredisposing polymorphism in Dpl. For example, detection may utilizestaining of cells or histological sections, performed in accordance withconventional methods. The antibodies of interest are added to the cellsample, and incubated for a period of time sufficient to allow bindingto the epitope, usually at least about 10 minutes. The antibody may belabeled with radioisotopes, enzymes, fluorescers, chemiluminescers, orother labels for direct detection. Alternatively, a second stageantibody or reagent is used to amplify the signal. Such reagents arewell known in the art. For example, the primary antibody may beconjugated to biotin, with horseradish peroxidase-conjugated avidinadded as a second stage reagent. Final detection uses a substrate thatundergoes a color change in the presence of the peroxidase. The absenceor presence of antibody binding may be determined by various methods,including flow cytometry of dissociated cells, microscopy, radiography,scintillation counting, etc.

[0167] If the particular disease state is associated with conformationaltransition of Dpl, diagnosis may rely on the recognition of thedisease-associated conformation of the protein. The disease-associatedform the protein may be distinguished from other forms of Dpl usingcharacteristics of the disease-associated conformation, e.g.insolubility, resistance to protease digestion, change in epitopeavailability and the like.

[0168] An alternative method for diagnosis depends on the in vitrodetection of binding between antibodies and Dpl in a lysate. Measuringthe concentration of Dpl binding in a sample or fraction thereof may beaccomplished by a variety of specific assays. A conventional sandwichtype assay may be used. For example, a sandwich assay may first attachDpl-specific antibodies to an insoluble surface or support. Theparticular manner of binding is not crucial so long as it is compatiblewith the reagents and overall methods of the invention. They may bebound to the plates covalently or non-covalently, preferablynon-covalently.

[0169] The insoluble supports may be any compositions to whichpolypeptides can be bound, which is readily separated from solublematerial, and which is otherwise compatible with the overall method. Thesurface of such supports may be solid or porous and of any convenientshape. Examples of suitable insoluble supports to which the receptor isbound include beads, e.g. magnetic beads, membranes and microtiterplates. These are typically made of glass, plastic (e.g. polystyrene),polysaccharides, nylon or nitrocellulose. Microtiter plates areespecially convenient because a large number of assays can be carriedout simultaneously, using small amounts of reagents and samples.

[0170] Patient sample lysates are then added to separately assayablesupports (for example, separate wells of a microtiter plate) containingantibodies. Preferably, a series of standards, containing knownconcentrations of normal and/or abnormal Dpl is assayed in parallel withthe samples or aliquots thereof to serve as controls. Preferably, eachsample and standard will be added to multiple wells so that mean valuescan be obtained for each. The incubation time should be sufficient forbinding, generally, from about 0.1 to 3 hr is sufficient. Afterincubation, the insoluble support is generally washed of non-boundcomponents. Generally, a dilute non-ionic detergent medium at anappropriate pH, generally 7-8, is used as a wash medium. From one to sixwashes may be employed, with sufficient volume to thoroughly washnon-specifically bound proteins present in the sample.

[0171] After washing, a solution containing a second antibody isapplied. The antibody will bind Dpl with sufficient specificity suchthat it can be distinguished from other components present. The secondantibodies may be labeled to facilitate direct, or indirectquantification of binding. Examples of labels that permit directmeasurement of second receptor binding include radiolabels, such as ³Hor ¹²⁵I, fluorescers, dyes, beads, chemiluminescers, colloidalparticles, and the like. Examples of labels which permit indirectmeasurement of binding include enzymes where the substrate may providefor a colored or fluorescent product. In a preferred embodiment, theantibodies are labeled with a covalently bound enzyme capable ofproviding a detectable product signal after addition of suitablesubstrate. Examples of suitable enzymes for use in conjugates includehorseradish peroxidase, alkaline phosphatase, malate dehydrogenase andthe like. Where not commercially available, such antibody-enzymeconjugates are readily produced by techniques known to those skilled inthe art. The incubation time should be sufficient for the labeled ligandto bind available molecules. Generally, from about 0.1 to 3 hr issufficient, usually I hr sufficing.

[0172] After the second binding step, the insoluble support is againwashed free of non-specifically bound material. The signal produced bythe bound conjugate is detected by conventional means. Where an enzymeconjugate is used, an appropriate enzyme substrate is provided so adetectable product is formed.

[0173] Other immunoassays are known in the art and may find use asdiagnostics. Ouchterlony plates provide a simple determination ofantibody binding. Western blots may be performed on protein gels orprotein spots on filters, using a detection system specific for Dpl asdesired, conveniently using a labeling method as described for thesandwich assay.

[0174] Other diagnostic assays of interest are based on the functionalproperties of Dpl proteins. Such assays are particularly useful where alarge number of different sequence changes lead to a common phenotype.For example, a functional assay may be based on the changes in thetranscriptional repertoire of cells mediated by addition of mediated byDpl gene products. Other assays may, for example, detect conformationalchanges, size changes resulting from insertions, deletions ortruncations, or changes in the subcellular localization of Dpl proteins.

[0175] In a protein truncation test, PCR fragments amplified from theDpl gene or its transcript are used as templates for in vivotranscription/translation reactions to generate protein products.Separation by gel electrophoresis is performed to determine whether thepolymorphic gene encodes a truncated protein, where truncations may beassociated with a loss of function.

[0176] Diagnostic screening may also be performed for polymorphisms thatare genetically linked to a predisposition for prion-mediated disorderssuch as CJD, particularly through the use of microsatellite markers orsingle nucleotide polymorphisms. Frequently the microsatellitepolymorphism itself is not phenotypically expressed, but is linked tosequences that result in a disease predisposition. However, in somecases the microsatellite sequence itself may affect gene expression.Microsatellite linkage analysis may be performed alone, or incombination with direct detection of polymorphisms, as described above.The use of microsatellite markers for genotyping is well documented. Forexamples, see Mansfield et al.,Genomics 24:225-233 (1994); Ziegle etal., Genomics 14:1026-1031 (1992); Dib et al., supra.

[0177] Microsatellite loci that are useful in the subject methods havethe general formula:

[0178] U (R)_(n) U′, where

[0179] U and U′ are non-repetitive flanking sequences that uniquelyidentify the particular locus, R is a repeat motif, and n is the numberof repeats. The repeat motif is at least 2 nucleotides in length, up to7, usually 2-4 nucleotides in length. Repeats can be simple or complex.The flanking sequences U and U′ uniquely identify the microsatellitelocus within the human genome. U and U′ are at least about 18nucleotides in length, and may extend several hundred bases up to about1 kb on either side of the repeat. Within U and U′, sequences areselected for amplification primers. The exact composition of the primersequences are not critical to the invention, but they must hybridize tothe flanking sequences U and U′, respectively, under stringentconditions. Criteria for selection of amplification primers are aspreviously discussed. To maximize the resolution of size differences atthe locus, it is preferable to chose a primer sequence that is close tothe repeat sequence, such that the total amplification product isbetween 100-500 nucleotides in length.

[0180] The number of repeats at a specific locus, n, is polymorphic in apopulation, thereby generating individual differences in the length ofDNA that lies between the amplification primers. The number will varyfrom at least 1 repeat to as many as about 100 repeats or more.

[0181] The primers are used to amplify the region of genomic DNA thatcontains the repeats. Conveniently, a detectable label will be includedin the amplification reaction, as previously described. Multiplexamplification may be performed in which several sets of primers arecombined in the same reaction tube. This is particularly advantageouswhen limited amounts of sample DNA are available for analysis.Conveniently, each of the sets of primers is labeled with a differentfluorochrome.

[0182] After amplification, the products are size fractionated.Fractionation may be performed by gel electrophoresis, particularlydenaturing acrylamide or agarose gels. A convenient system usesdenaturing polyacrylamide gels in combination with an automated DNAsequencer, see Hunkapillar et al., Science 254:59-74 (1991). Theautomated sequencer is particularly useful with multiplex amplificationor pooled products of separate PCR reactions. Capillary electrophoresismay also be used for fractionation. A review of capillaryelectrophoresis may be found in Landers, et al., Bio Techniques14:98-111 (1993). The size of the amplification product is proportionalto the number of repeats (n) that are present at the locus specified bythe primers. The size will be polymorphic in the population, and istherefore an allelic marker for that locus.

[0183] Diagnosis and therapeutic treatment of PrP-associated disorders

[0184] Animals with increased expression of Dpl activity, such as thetwo PrP^(0/0) lines described herein, also display a reduced incubationperiod for development of the symptoms of prion infection. Based on thisobservation, the present invention provides assay methods fordetermining the prion infectivity of a sample using animals that have analtered expression of Dpl. The method determines whether a sample isinfected with prions by inoculating a Dpl transgenic or hybrid mammal ofthe invention with a sample to be tested and observing the mammal for aperiod of time sufficient to determine if they develop symptoms of adisease normally associated with prions. The animals may have anincreased level of Dpl expression due to 1) a natural endogenous Dplmutation, e.g. a mutation in the Dpl locus identified in a geneticscreen; 2) a Dpl mutation produced in a transgenic animal, e.g. adeletion of the PrP locus that results in increased expression of theDpl gene product; and/or 3) the introduction of an exogenous transgenethat encodes a Dpl polypeptide.

[0185] The Dpl animals of the present invention can also be used in amethod for determining the cause of death of an animal. Such a methodinvolves inoculating a Dpl transgenic or hybrid animal of the inventionwith body fluid or tissue such as extracted brain tissue from the animalwhich has died (and preferably inoculating control animals with astandardized preparation of prions) and observing the transgenic orhybrid animal (and control animals) in order to determine if the animaldevelops symptoms of prion infections.

[0186] In a preferred embodiment, the animals of the invention withaltered Dpl activity also have a genome that is altered with respect tothe PrP locus. Exemplary animals include, but are not limited to: (1)animals with ablated endogenous PrP genes, as disclosed in U.S. Pat. No.5,698,763; (2) animals with an ablated endogenous PrP gene and anexogenous PrP transgene from a genetically diverse animal, as disclosedin U.S. Pat No. 5,792,901 and U.S. Ser. No. 08/935,363; and (3) animalswith an ablated endogenous PrP gene and an inducible PrP transgene, asdisclosed in U.S. Ser. No. 09/052,963

[0187] Preferred host animals are mice and hamsters, with mice beingmost preferred in that there exists considerable knowledge on theproduction of transgenic animals. Other possible host animals includethose belonging to a genus selected from Mus (e.g. mice), Rattus (e.g.rats), Oryctolagus (e.g. rabbits), and Mesocricetus (e.g. hamsters) andCavia (e.g. guinea pigs). In general mammals with a normal full grownadult body weight of less than 1 kg which are easy to breed and maintaincan be used. The host PrP gene can be changed to include codons fromgenetically diverse PrP genes from test animals belonging to a genusselected from Bos, Ovis, Sus and Homo. Preferably, a mouse host PrP geneis changed to include codons from a human, cow or sheep PrP gene, withcow being most preferred. Cows are preferred because an important objectof the invention is to use the animal to test a statisticallysignificant number of cows in a herd of cows to determine if the cowsare infected with prions which cause BSE, known as “mad cow” disease.

[0188] The present invention also provides a method of testing theefficacy of a drug in the treatment of disease developed as a result ofinfection with prions comprising administering a drug to be tested to atransgenic or hybrid animal infected with prions (preferably astandardized prion preparation) and observing and/or testing the mammalto determine if the drug aids in treating or slowing the progress of thedisease or its symptoms. Such methods are described in U.S. Pat No.5,792,901 and U.S. Ser. No. 08/935,363, which are both incorporatedherein by reference for this purpose.

[0189] The present invention also provides a method for treating priondisease by administration of a compound that downregulates Dplexpression and/or decreases Dpl activity. Since increased Dpl activityis associated with a reduction in the incubation period for priondisease, and since PrP^(c) can rescue the increased Dpl phenotype(suggesting they may either interact or compete in a pathway),decreasing Dpl activity can slow or halt the progression of priondisorder, for example by prevention of the degeneration of neurons inresponse to PrP^(Sc).

[0190] Therapeutic Uses of Dpl-Encoding Nucleic Acid

[0191] Dpl-encoding nucleic acid can be introduced into a cell toaccomplish transformation of the cell, preferably stable transformation.Introduction of the Dpl-encoding nucleic acid into the cell can beaccomplished according to methods well known in the art (e.g., throughuse of electroporation, microinjection, lipofection infection with arecombinant (preferably replication-deficient) virus, and other meanswell known in the art). Preferably, the Dpl-encoding nucleic acid isoperably linked to a promoter that facilitates a desired level of Dplpolypeptide expression (e.g., a promoter derived from CMV, SV40,adenovirus, or a tissue-specific or cell type-specific promoter).Transformed cells containing the Dpl-encoding nucleic acid can beselected and/or enriched via, for example, expression of a selectablemarker gene present in the Dpl-encoding construct or that is present ona plasmid that is co-transfected with the Dpl-encoding construct.Typically selectable markers provide for resistance to antibiotics suchas tetracycline, hygromycin, neomycin, and the like. Other markers caninclude thymidine kinase and the like.

[0192] The ability of the transformed cells to express the Dpl-encodingnucleic acid can be assessed by various methods known in the art. Forexample, Dpl expression can be examined by Northern blot to detect mRNAwhich hybridizes with a DNA probe derived from the relevant gene. Thosecells that express the desired gene can be further isolated and expandedin in vitro culture using methods well known in the art. The host cellsselected for transformation with Dpl-encoding DNA will vary with thepurpose of the ex vivo therapy (e.g., Dpl peptide production), the siteof implantation of the cells, and other factors that will vary with avariety of factors that will be appreciated by the ordinarily skilledartisan.

[0193] Methods for in vivo delivery of a nucleic acid of interest forexpression in a target cell are known in the art. For example, in vivomethods of gene delivery normally employ either a biological means ofintroducing the DNA into the target cells (e.g., a virus containing theDNA of interest) or a mechanical means to introduce the DNA into thetarget cells (e.g., direct injection of DNA into the cells, liposomefusion, pneumatic injection using a “gene gun,” and the like).

[0194] The amount of DNA and/or the number of infectious viral particleseffective to infect the targeted tissue, transform a sufficient numberof cells, and provide for production of a desired level of Dpl can bereadily determined based upon such factors as the efficiency of thetransformation in vitro and the susceptibility of the targeted cells totransformation. Generally, the amounts of DNA can be extrapolated fromthe amounts of DNA effective for delivery and expression of the desiredgene in an animal model. For example, the amount of DNA for delivery ina human is roughly 100 times the amount of DNA effective in a rat.

[0195] Regardless of whether the Dpl-encoding DNA is introduced in vivoor ex vivo, the DNA (or cells expressing the DNA) can be administered incombination with other genes and other agents. In addition, Dpl-encodingDNA (or recombinant cells expressing Dpl DNA) can be usedtherapeutically for disorders associated with, for example, a decreasein Dpl production, but which are not associated with an alteration inDpl function per se.

EXAMPLES

[0196] The following examples are put forth so as to provide those ofordinary skill in the art with a complete disclosure and description ofhow to make and use the present invention, and are not intended to limitthe scope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

EXAMPLE 1 Identification of the Prnd Locus

[0197] Previous large-scale sequencing studies of phage and cosmidmolecular clones encompassing human, sheep, and the “a” allele of themouse PrP gene, Prnp^(a), failed to reveal any evidence of additionalcoding regions, either immediately adjacent to PrP or within intronicsequences (Lee et al., Genome Res. 8:1022-1037 (1998)). However,sequencing of the mouse Prnp^(b) cosmid clone I/LnJ-4 (Westaway et al.,Neuron 7:59-68 (1991)), which extends further in a 3′ direction andanalysis with the “XGrail 1.3 c” program (Uberbacher et al, ORNL/TM11741 (1991)) revealed a candidate coding region at nt 36119 to 36,751(FIG. 1A).

[0198] Cosmids and YAC clones were propagated by standard procedures andhave been described previously (D. Westaway, Cell 76:117-29 (1994)). A129Sv/J BAC clone 321L17 was identified from a library kindly providedby Millenium Pharmaceuticals.

[0199] The predicted amino acid sequence of this ORF (FIG. 2) exhibitshomology to mammalian, marsupial and avian PrP. We refer to the putativegene as Prnd (prion gene complex, downstream) and the protein product as“Dpl” downstream, prion protein-like: German, “double”)

[0200] Southern blot analyses indicated that the putative coding regionof Prnd was a single-copy sequence in the mouse genome. To exclude thePrnd ORF as an idiosyncrasy of I/LnJ mice, we sequenced thecorresponding chromosomal region from a C57B6-derived YAC clone(Westaway et al., Proc. Natl. Acad. Sci. USA 91:6418-6422 (1994)) and a129/SvJ BAC clone 321L17. Both revealed an identical ORF. Furthermore,related ORFs are present in rats and humans, with the human PrnD genepositioned 27 kb 3′ to PRNP. These data indicate that PrnD genes will befound to be present in all mammals.

EXAMPLE 2 Structure of the Prnd Locus

[0201] Prnd cDNAs were isolated to demonstrate the splicing of mRNAstranscribed from this region and to define appropriate coding exons. Thecomplimentary approaches of cDNA library screening and rapidamplification of cDNA ends (RACE) were undertaken using wild type adultmouse brain cDNA as starting material.

[0202] RACE cloning of PrnD RNAs

[0203] For 5′ RACE analysis, “Marathon” mouse brain cDNA (Clontech, PaloAlto, Calif.) was amplified with adapter primer AP1 and the Prndanti-sense strand primer DW112, 5′-CGGTTGGTCCACGGCGACCCGAA-3′(0.2 FM),using a Perkin-Elmer 2400 thermocycler and “touchdown” PCR conditions of94 EC 20 seconds, 5 cycles of 94 EC, 5 sec and 72 EC, 360 seconds; 5cycles of 94 EC, 5 seconds and 70 EC 360 seconds; and 30 cycles of 94EC, 5 seconds and 68 EC, 360 seconds. Taq polymerase was used inconjunction with “TaqStart” antibody (Clontech, Palo Alto, Calif.).Size-fractionated reaction products were resuspended in Tricine bufferand a second-round of PCR was performed using a nested primer set: AP2universal primer and the Prnd primer ORFP-R2 (0.2 FM),5′-GCAGATCTCTTTGATCAGCC-3′ (94 EC, 60 seconds, and 20 cycles of 94 EC 10seconds, 57 EC, 20 seconds, 72 EC 150 seconds. Subsequent toverification by hybridization to a kinase-labeled internal primer DW955′-CAGATCCACCGAAGCTCGGG-3′, PCR reaction products were either sequenceddirectly or subsequent to cloning into the plasmid vector pCR2.1 TOPO(Invitrogen). A similar strategy was adopted for cloning of 3′ RACEproducts, using the nested primers

[0204] DW111, 5′-TGGTGACCAGCTGCGTCAACGCCA-3′ and

[0205] DW192, 5′-TGGGAAGGCCCTGAGCGACAACCGTG-3′.

[0206] RT-PCR of Prnd mRNAs

[0207] Total RNA was prepared by the acid-phenol method andpolyA-selected on oligo dT-latex (Quiagen). First-strand cDNA synthesiswas carried out using 10 ng of polyA+RNA primed with oligo dT or randomhexamer primers (as noted) and MuLV reverse transcriptase, asrecommended by the manufacturer (Stratagene, La Jolla, Calif.). 2 ngaliquots of cDNA synthesis reactions incubated plus or minus reversetranscriptase were either amplified directly, or following concentrationand de-salting by ultrafiltration. In the latter instance, a cDNAfirst-strand synthesis reaction of 40 ul (equivalent to 160 ng cDNA) wasdiluted with 450 ul water and introduced into a 50,000 molecular weightcut-off “Eluticon” microconcentrator (Owl Scientific). Subsequent tospinning at 14,000 xg, the membrane was washed with 500 ul water, spun,inverted and eluted with 10 ul water. 2.5 ul of this preparation wasused per PCR reaction. Alternatively, 10 ug of total RNA wasreverse-transcribed in a total volume of 50 ul using Superscript reversetranscriptase (Life Technologies) with 2.5 ul used per amplificationreaction. “Hot-start” amplification (“Platinum Taq”, Life Technologiesor Advantage KlenTaq, Clontech) was performed in standard reactionbuffers, as noted below. Primer sets were as follows:

[0208] Prnd exon 1 a to PrnD exon 2:

[0209] DW189′ 5′-GCTCCAAGCTTCAGAGGCCACAGTAGCA-3′ and

[0210] DW96, 5′-TTACTTCACAATGAACCAAACGAAAC-3′ (1 uM):

[0211] 94 EC, 180 seconds; 40 cycles of 94 EC, 15 seconds; 65 EC 30seconds; 72 EC, 75 seconds, using Platinum Taq polymerase and MgCl₂ at afinal concentration of 2 mM.

[0212] Intergenic exon 2 to PrnD exon 2:

[0213] DW117, 5′-GAGTGGAGGTCTTCGCGCA-3′ and DW96 (0.5 uM).

[0214] 95 EC 300 seconds, and 45 cycles of 95 EC, 10 seconds, 55 EC 20seconds, 72 EC 150 seconds, using Platinum Taq polymerase, and MgCl₂ ata final concentrations of 2.5 mM.

[0215] Prnp exon 2 to PrnD exon 2:

[0216] 5′UT.3 (Westaway, Neuron 7:59-68 (1991)) and DW96 (0.2 uM).

[0217] 95 EC 300 seconds, and 40 cycles of 94 EC, 15 seconds, 60 EC 30seconds, 72 EC 120 seconds, using Platinum Taq polymerase and MgCl₂ andDMSO at a final concentrations of 2.5 mM and 4% respectively.

[0218] Rat Prnp exon 2 to the 3′ end of PrnD (defined by ESTAI136375):

[0219] DW213 5′-TCAAAACTGAACCATTTCAACCCAACTGAAGTATTCTGCC-3′and

[0220] DW214 5′-ACCCAGCGTTCTGGCCCGGTATTAGGATT-3′ (mismatches to themouse gene sequences are underlined). 94 EC for 120 seconds followed by40 cycles of 94 EC, 15 seconds, and 72 EC 240 seconds. β-Actin: thesewere used as recommended by the manufacturer (Statagene, La Jolla,Calif.).

[0221] Results from the RACE and RT-PCR analyses were used to search forEST matches from public databases and a number were retrieved frommouse, human and rat (FIG. 1B). PrnD cDNAs of 1.5 and 1.7 Kb wereretrieved from a Balb/C testis cDNA library. These clones correspondedto the major Dpl mRNA species observed on Northern blots. A largertranscript of 2.7 Kb was also apparent on northern blots and 3′ RACEprovided good evidence for alternative splicing of exon 2, which couldaccount for this mRNA species.

[0222] Examination of the two major cDNA clones indicated that they werenearly full length, as they contained polyA tracts at the 3′ end andpartial sequence from alternately spliced 5′ exons. 5′ RACE analysis wasin close agreement with the sequence of these clones indicating splicingevents from short 5′ exons, denoted exons 1 a and 1 b respectively,starting at either nt 34,124 or nt 34,277 to a common splice acceptorlying immediately 5′ to exon 2, the Prnd ORF, at nt 36,204 (FIG. 1B,FIG. 2A, 2B). This was also in agreement with the sequence of a mouseDpl EST AA796652, which is generated by splicing of exon 1 b to exon 2(FIG. 1B). The 5′ boundary of the PrnD 5′ untranslated region exon waslocated within an interval of approximately 30 nucleotides by RT-PCRreactions with different primers (FIG. 2A). Primer extension reactionsand nuclease protection assays were employed for more precise mapping.Primer-extension reactions with primers DW197,(5′-CCAGCCGGTTCTTCATGGTGAATCTCGG-3′, hybridization temperature 65 EC),and DW123 a, (5′-CATGGTGAATCTCGGTTCTC-3′, hybridization temperature 55EC), were carried-out as before (Westaway et al., 1987) except thatprimers were labeled to a specific activity of

9×10⁷ dpm/pmole using 6000 Ci/mmol gamma ³² p -ATP (“Kinase Max”, AmbionInc: NEN). A 42-mer oligonucleotide labeled in the same manner was usedfor a nuclease protection assay (“multi--NPA”, Ambion Inc.).

[0223] Results of these mapping experiments were in close agreement,defining a cluster of mRNA start-sites 13-18 nucleotides upstream of 5′termini defined by RACE cDNAs and EST gbAA796652 (from mouse mammarygland). These assignments are also in accord with the structure of humanPrnd cDNAs. Because of the alternative 3′ boundaries of this 5′non-coding exon, arising from the use of two alternative splice donors,it is denoted “exon 1 a /1 b ”. Notably, a similar arrangement has beenobserved in the bovine PrP gene (Horuichi et al., Biochem. Biophys. Res.Commun. 233:660-654 (1997)). Since neither exon 1 a or 1 b contains ATGcodons in-frame with the chromosomal ORF, these cDNAs (and othersdescribed below) indicate that the initiation codon for Prnd lies 3′ tothe splice acceptor at nucleotide 36,212. This ATG is conserved andconforms closely to the Kozak consensus for the initiation of eukaryoticmRNAs (consensus GCCGCCa/gCCATGG; PrnD AGATTCACCATGA) (Kozak, NucleicAcids Res. 15:8125-8148 1987). The position of the PrnD start site isorganized in a similar fashion to Prn-p itself, where the ATG codon lies10 nucleotides 3′ to the splice acceptor of exon 3 (Westaway et al.,Cell 51:651-662 (1987)).

[0224] The internal structures of independently-derived cDNAs indicatedthat the Prnd mRNA 3′UTR is encoded within separate exons and subject toalternative splicing (FIG. 1B). Thus one splice donor 28 nucleotides 3′of the ORF termination codon (position 36,779) codon and another furtherdownstream at 37,472 supply a splice acceptor at position ˜38,006,suggesting an explanation for the ˜1.7 and 2.7 kb RNAs detected byNorthern blots (below). cDNA clones containing polyA tracts were foundto terminate at positions 39,099 and 39,315 in the I/LnJ-4 cosmid. Whilethe more 3′ site is preceded by a consensus polyadenylation signal,AATAAA, a similar motif is lacking from the 5′ site. Of note, a sequenceCTTAAA is located 27 nucleotides 5′ of the polyA tail. This 3′ genearchitecture is unusual as (i) introns within 3′UTR coding sequences arerare, (ii) splice sites at positions 37,472 and 38,006 differ from theaccepted consensus (such that definitive positioning of the exact intronboundaries are complicated by nucleotide redundancies whereby theboundary may be shifted by +1 or +2 from these coordinates) or a strainpolymorphism, and (iii), intron 2 defines the putative protein codingexon of Prnd as being an internal, rather than a terminal exon, as inthe case of Prnp. With a size of either 575 or 1,268 nucleotides(depending upon the selection of splice donor site) this exoncontravenes the rule that 99% of internal exons are less than 400 basesin length (Berget, J. Biol. Chem. 270:2411-2414 (1995)).

EXAMPLE 3 Expression of Transcripts from the Prnd Locus

[0225] Northern blot analyses were performed using the entire 570 bpPrnD ORF fragment derived from the ILn/J-4 cosmid using total (50 gloading) or oligo-dT selected RNA (7 g loading). Total RNA was madeusing Trizol reagent (Gibco) following the manufacturers instructions.Poly A+ RNA was isolated using a Oligotex mRNA kit (Qiagen). RNA sampleswere heated for 30 minutes to 50 deg C. in glyoxal sample buffer andelectrophoresed on 1.2% agarose gels as recommended by the manufacturer(Ambion Inc., Woodland, Tex.). RNA was transferred onto Hybond N+(Amersham) in 5 XSSC 10 mM NaOH for 2 hours using a turboblotter(Schleicher and Schuell), rinsed in 5 XSSC, UV fixed (Statagene, LaJolla, Calif.) then prehybridized (6 xSSC, 1% SDS, 3% (w/v) dextransulfate, 10 ug/ml sonicated herring sperm DNA) for 1 hour at 65 EC priorto the addition of a 540 bp Dpl ORF PCR a-dCTP³² random-primed probe(Feinberg et al., Anal. Biochem. 132:6-13 (1983)) generated with theprimers RM1 (5′-ATGAAGAACCGGCTGGGTAC-3′) and DW96 with 129Sv/J genomicDNA template. Membranes were hybridized for 16 hours then rinsed for 5minutes at room temperature in 2XSSC/1%(w/v) SDS, washed 30 minutes at65 deg C. in 2XSSC/1%(w/v) SDS then 30 mins in 1 XSSC/1%(w/v) SDS at 65deg C. Blots were exposed at −80 deg C. for 3 days. This revealed PrndmRNAs of approximately 1.7 and 2.7 kb in testis and heart polyA+ RNA ofwild type mice. No Prnd mRNA was detected in brain.

[0226] Using RT-PCR, mRNA in wild type mice produced by splicing fromexon 1 a to exon 2 could be detected in adult brain cDNA and 14-dayembryos (FIG. 3). Additional cDNA species, one of which reflectsinclusion of exon 1 b, were detected in some adult brain samples. Somepreliminary evidence of Dpl mRNA expression can be derived from thetissue origin of the Prnd EST matches (FIG. 1B): mouse ESTs AA796652,AA104098 and AA190150 were derived from adult mammary gland, embryonicheart and adult spleen respectively. In sum, Prnd mRNAs are expressed inembryos, in peripheral tissues, and at low levels in the adult CNS.

[0227] As predicted from the DNA sequence data, species of similarelectrophoretic mobility were apparent in brain RNA of Tg(MoPrP-B)2091mice, harboring a high copy-number array of an ILn/J-4 cosmid transgenewithin which Dpl was first noted. A corresponding product wasundetectable in wt control mouse brain samples as noted above.

EXAMPLE 4 Computer Analysis of Dpl Sequences

[0228] Various non-redundant combinations of the SWISSPROT and TREMBL(A. Bairoch et al., Nucleic Acids Res. 19 Suppl:2247-9 (1991); Stoesser,et al., Biochemistry 37:7185-7193. (1998)) sequence databases weresearched with the sequences of human and mouse Dpl using the alignmentprograms FASTA_(—)3 (Pearson et al., Proc. Natl. Acad. Sci. USA85:2444-2448 (1988)) and PSI_BLAST (S. F. Altschul et al., Nucleic AcidsRes.25:3389-402 (1997). The Dpl proteins show distant but significanthomology (24-26% identity over ˜160 residues) to many PrP sequences(FASTA_(—)3 score E '0.00015, where E is the expected number ofsequence matches by chance; PSI-BLAST E '2×10(−41) for alignment withdomestic dog PrP).

[0229] Possible transmembrane segments were predicted using TopPred 2(G. von Heijne, J Mol Biol. 225:487-94 (1992)) and TMPred (See e.g. D.S. Millican et al., Endocr Res. 24:387-90 (1998). N-terminal signalpeptide cleavage sites for human and MoDop were predicted to be at thepositions indicated using the SignalP program (H. Nielsen, Int J NeuralSyst. 8:581-99 (1997)). This program locates eukaryotic signal peptideswith ˜70% accuracy. However, 50% of Type-TI N-terminal transmembranesegments are over-predicted as signal peptides. The GPI attachment site(denoted w) and the succeeding two residue positions (w+1 and w+2) havedistinct residue preferences and aversions, e.g. preferences for serineand aspartic acid at position w (Y. Furukawa Biochim Biophys Acta1328:185-96 (1997); P. Harrison, unpublished data) and requires astretch of mostly hydrophobic residues (8-31 residues long) in thecleaved C-terminal signal peptide. This region is separated from w+2 bya 2-8 residue segment of greater hydrophilicity. Both signals are foundin the Dpl sequences. The most likely GPI-anchor attachment site for Dpl({w, w+1, w+2 }′ G 125A(G or A) ) was determined from an examination ofthe literature on GPI anchors, the sequence homology between the Dplsand PrPs at the C-terminus and from a representative set of GPI-anchoredproteins culled from sequence databases (P. Harrison, unpublished data)(FIGS. 2 and 5. The GPI anchor site suggested by the multiple sequencealignment ({w, w+1, w+2 } 'G157LR) is unlikely (FIG. 2) as w+1 is almostnever a large aliphatic hydrophobic or aromatic (97%; 61/63 examples)and w+2 is almost never charged (or large aliphatic hydrophobic oraromatic) (98%; 62/63) (P. Harrison, unpublished data). Other possibleattachment sites in this region can be discounted for similar reasons,leaving only the suggested site, {w, w+1, w+2 } ′ G155A(G or A).

EXAMPLE 5 Chimeric Prnp/PrnD mRNAs

[0230] In parallel to RACE and cDNA cloning, the gene-finding program“GRAIL” was also used to identify possible 5′ exons of PrnD. Analyses ofthe relevant area of the I/LnJ-4 cosmid sequence did not detect exon 1 a/1 b, but instead two putative sense-strand exons (nt 29,589-29,672;“Grail exon 6” and nt 29,798-29,832 ; “Grail exon 7”) between the Prnpand PrnD ORFs. Using a primer located in “Grail exon 6”, cDNA could beamplified from adult wild-type mouse brain RNA, with DNA sequencingconfirming splicing from a consensus splice donor at nucleotide 29,671to the PrnD exon 2 splice acceptor (FIG. 4).

[0231] These RT-PCR analyses defined grail exon 6 as a new 5′ exon (FIG.4, intergene exon 2), but our failure to identify cDNAs including PrnDexon 1 a /1 b (or “Grail exon 7”) suggested that conserved sequences 5′to exon 1 a constitute the PrnD promote proper. The possibility that thePrnD mRNAs including “Grail exon 6” originate from a more 5′ promoterwas then examined. Since the Prnp promoter is located only 23 Kb awayand positioned in the correct transcriptional orientation, this was astrong candidate for the PrnD promoter as well. Using a 5′ primerlocated in the Prnp exon 2 and a 3′ primer in PrnD exon 2, a product wasobtained in RT-PCR analyses of brain cDNA from wild-type mice (FIG. 4).Sequence analysis of these cDNAs confirmed their chimeric nature, thatis, starting within the Prnp transcription unit at exon 2 and finishingwithin the PrnD transcription unit commencing at exon 2 a (FIG. 5). SomecDNAs included the “Grail 6” exon interposed between Prnp exon 2 andPrnD exon 2 a and thereby defined the 5′boundary of this exon as aconsensus splice acceptor site at nucleotide 29,671. Other cDNAsincluded an additional exon (intergene exon 2), located betweennucleotides 25,482 and 26004, in a sub-set of cDNAs. Alternative use ofthese two intergene exons results in a variety of chimeric mRNAs (FIG.5).

[0232] Since there is only one precedent for intergenic splicing in thechromosomal genes of a higher mammal (Magrangeas et al., J. Biol. Chem.273:16005-16010 (1998)), we sought to confirm our observations in asecond species. Using the sequence of rat Prnp exon 2 and EST AI136375homologous to the 3′ boundary of mouse PrnD exon 3, RT-PCR experimentswere performed for rat tissues.

[0233] As predicted, analogous chimeric cDNAs including two permutationsof intergene exons were detected in both brain and testis. Since the twointergene exons are poorly conserved between mouse and rat (notpresented) and fail to exhibit ATG codons in-frame with the PrnD ORF, weinfer that they do not serve a protein coding function.

EXAMPLE 6 Establishment of Cell Lines Expressing Dpl

[0234] In order to better study the activity and/or cellularinteractions of Dpl, a Dpl transgene was transfected into an establishedneuronal cell line.

[0235] Preparation of mammalian Dpl-expression vector.

[0236] A PrnD cDNA cassette was prepared by amplification of the B6-9YAC clone with the oligonucleotide primer pair

[0237] DW174′ 5′-CGGAATTCCAGCCTTTCCCTTGCCGATTCAC-3′ and

[0238] DW175′ 5′-GCTCTAGAACTGGGCTACCTCTGTCTACCT-3′. The 5′ primer ofthis pair alters the exon 2 a splice acceptor site. Subsequent todigestion with EcoR1 and Xba the resulting cDNA cassette wasgel-purified and cloned into the mammalian expression vector pcDNA3.0(Invitrogen).

[0239] Establishment of stable Dpl cell lines.

[0240] pcDNA3.0 _Dpl (1-5 g) was added to 0.1 mL Opti-MEM (LifeTechnologies), mixed with 15L LipofectAMINE reagent (Life Technologies)and then incubated at room temperature for 40 minutes. Neuro-2 a cells[N2 a] (ATCC), cultured in high glucose DMEM (Life Technologies)supplemented with 10% fetal bovine serum [FBS] (Life Technologies), weregrown to 60% confluency in 6 cm dishes. Cells were washed twice withHank's balanced salt solution [HBBS] and 2 mL Opti-MEM was added to eachdish. The lipid/DNA mixture was added dropwise to each dish with gentlemixing and cells were incubated for 5 hours at 37 EC. Cells were alsotransfected with pcDNA3.0 lacking the PrnD insert, and Opti-MEM/lipidmixture containing no DNA as negative controls. Cells were then fed with1 mL DMEM containing 20% FBS and incubated overnight at 37 EC.Trypsinized cells and seeded 10 cm plates with DMEM containing 10% FBSand 1 mg/mL G418. Individual colonies were selected and expanded after 2weeks of selection, and maintained in DMEM containing 10% FBS and 0.3mg/mL G418.

[0241] Preparation of RNA from Cell Lines.

[0242] Cells were grown to 80% confluency in 75 cm flasks, washed threetimes in 12 mL HBBS. Cells were scraped into 5 mL ice-cold HBBS andpelleted at 1,000 x g for 5 min at 4 EC. Cells were resuspended in 1 mLice-cold HBBS, transferred to a RNA-free microfuge tube (Ambion) andpelleted at 14,000 rpm for 1 minute at 4 EC. The pellet was resuspendedin 0.6 mL freshly-prepared denaturing solution (4M guanidiniumthiosulphate, 25 mM sodium citrate, pH 7.0, 0.5% Sarkosyl, 0.1Mβ-mercaptoethanol). Next 60L 2M sodium acetate, pH4.0, 0.6 mL acidphenol, and 0.13 mL chloroform: isoamyl alcohol (24:1) were added andthe contents of the tube were mixed vigorously by shaking. The samplewas incubated on ice of 20 min and then spun at 14,000 rpm for 20minutes at 4 EC. The aqueous phase was transferred to a new RNA-freemicrofuge tube and 0.6 mL ice-cold isopropanol was added. The tube wasmixed by shaking and incubated at −20 EC for 1 hr. The tube was thencentrifuged at 14,000 rpm at 4 EC for 20 min and the isopropanol wasremoved. The pellet was washed in 75% ethanol, made up usingDEPC-treated ddH₂O, and incubated at −20 EC for 20 min. The tube wasthen spun at 14,000 rpm at 4 EC for 20 min and the ethanol was removed.After air-drying, the pellet was resuspended in 30L DEPC-ddH₂O, 1L wasin order to determine the RNA concentration by absorbence at 260 nm, andthe sample was immediately snap frozen on dry-ice and stored at −80 EC.

[0243] Northern Blot Analysis of Dpl Cell Lines

[0244] RNA samples were thawed on ice and brought to 2 g/L usingDEPC-ddH₂O. 5L of each sample was analyzed. Total RNA was fractionatedon a 1% agarose gel containing formaldehyde using the NorthernMax kit(Ambion). The gel was then transferred to Nytran-plus membrane (S&S)also using the NorthernMax kit. A 400 bp Dpl ORF fragment was preparedby PCR amplification of the PrnD cDNA cassette using primers DW95 andDW96, and random primed using the DECAprime II kit (Ambion). Themembrane was hybridized using ExpressHyb solution (Clontech) and exposedto X-ray film at −80 EC for 72 hr.

EXAMPLE 7 Dpl Regulation and Ataxic Behavior in Prnp^(0/0) Mice.

[0245] The alteration of Prnd expression in Prnp^(0/0) lines wasdetermined to be responsible for the ataxia observed in two PrP knockoutlines of mice lines (Sakaguchi et al., Nature 380:528-531 (1996)). cDNAsprepared from different lines of Prnp^(0/0) mice were shown to behavedifferently in semi-quantitative RT-PCR analyses. The differencescorresponded with both the structure of the ablated allele design andthe PrP deficient phenotype. The structures of the 4 independentlygenerated alleles is shown in FIG. 6, and fit into two main classes:those that create in internal insertion/deletion within PrP exon 3(Büeler et al., Nature 356:577-582 (1992); Manson et al., Mol.Neurobiol. 8:121-127 (1994)), which do not develop a late-onset ataxia,and two alleles which remove the entire PrP ORF but also a˜1 Kb region5′ to exon 3, including the exon 3 splice acceptor site. Thus ZrchPrnp^(0/0) mice (Büeler et al., Nature 356:577-582 (1992)) with anintact splice acceptor gave no signal while the Rcm and Ngsk lines ofPrnp^(0/0) mice with an ablated exon 3 splice acceptor site showed apotent increase in chimeric mRNA relative to wild-type mice. Control PCRreactions performed with alternative primer sets were used to excludetrivial differences between cDNA preparations.

[0246] Northern blot analyses showed that the expression levels of DplmRNA in brain increases dramatically in the two Prnp^(0/0) linesdeveloping ataxia. Thus, overexpression of PrnD mRNA in brain isassociated with the development of ataxia in Ngsk and Rcm Prnp^(0/0)mice while the low levels of PrnD mRNA in brains of Zrch Prnp^(0/0) miceis not accompanied by cerebellar dysfunction. The following tablesummarizes these results: TABLE 1 Cerebellar ataxia and Dpl expressionin lines of Prnp^(0/0) mice Ataxia and Deletion of exon cerebellar 3splice acceptor Overexpression degeneration in in Prnp of PrnD mRNAPrnp^(0/0) line aged mice null allele in brain Zrch absent no no Npuabsent no ND Ngsk present yes yes

[0247] Profound overexpression of Prnd occurs in Ngsk and Rcm lines ofPnrp^(0/0) mice, both of which develop ataxia. Equally striking is thefinding that Purkinje cells remain healthy in Zrch Pnrp^(0/0) mice,which do not overexpress Prnd. These findings suggest thatoverexpression of Prnd is toxic for Purkinje cells. Because expressionof chimeric mRNAs is dependent upon the Prnp promoter, it is of interestto note that this promoter is active in Purkinje cells (Nishida et al.,in press), with a putative Purkinje cell-specific enhancer elementinferred to exist either within Prnp intron 2 or 3′ of a Sal Irestriction site in the Prnp/Prnd intergene region (Fischer et al., EMBOJ. 15:1255-1264 (1996)).

EXAMPLE 8 Predicted Features of the Dpl Proteins

[0248] Comparison of available Dpl peptide sequences reveals a proteinwhich is highly conserved, providing evidence that Dpl is a functionalgene product under selection pressure (FIGS. 7 and 8). Mouse and rat Dplproteins share >90% identity whilst mouse with the human Dpl ORF reveals76% sequence identity. The majority of the sequence differences betweenmouse and human (94%, 32/34) are conservative amino acid replacements.Homology between Dpls and mouse PrP are estimated at between 20 and 24%,slightly lower than that observed between chicken and mammalian PrPs(31-34%) (Gabriel et al., Molecular cloning and structural analysis of acandidate chicken prion protein. In Prion Diseases of Humans andAnimals, S. B. Prusiner, J. Collinge, J. Powell and B. Anderton, eds.(London: Ellis Horwood), pp. 407-431 (1992); Harris et al., Proc. Nat.Acad. Sci USA 88:7664-7668 (1991)). Dpls from all three species have apredicted N-terminal signal peptide cleavage site indicating that theyare synthesized, like PrP^(c), in the secretory pathway (FIG. 9). PrPhas a cluster of basic residues rich in arginine and lysine adjacent toa predicted N-terminal signal peptide cleavage site and Dpl shares asimilar feature between residues 25 to 38 in which 7 residues (50%) arebasic.

[0249] Dpl lacks a convincing His/Pro/Gly rich octarepeat region whichis present in PrP and which is thought to bind Cu(II) ions in vivo(Brown et al., Nature 390:684-687 (1997); Stöckel et al., Biochemistry37:7185-7193 (1998); Viles et al., Proc. Natl. Acad. Sci. USA96:2042--2047). However, mouse Dpl has a short PSSGGQ motif betweencodons 41-46 very similar to a PSSGGS motif in chicken PrP codons103-108. The significance of this observation is uncertain because weare unsure whether Dpl or PrP is the original molecule: i.e. whether Dplwas generated from a recent gene duplication of PrP or vice versa.Either way, it could be argued that this motif represents a vestigialrepeat which has been deleted or, alternately, a prototypic unit whichhas been amplified to form the repeat structure within PrP.

[0250] Dpl lacks a region with significant homology to the AGAAAAGAmotif completely conserved in all known PrP sequences and correspondingto mouse PrP codons 112-119 (Bamborough et al., Cold Spring Harb. Symp.Quant. Biol. 61:495-509 (1996); Schätzl et al., J. Mol. Biol.245:362-374 (1995)). This difference may indicate a significantfunctional divergence between Dpl and PrP because this region of PrP hasbeen shown to be critical for PrP topology in the ER membrane (Hegde etal., Science 279:827-834 (1998)) and the synthetic PrP peptide 106-126has also been shown to be neurotoxic to primary cultures exposed to thesynthetic peptide 106-126 (Forloni et al., Nature 362:543-546 (1993)).

[0251] PrP has a glycosylphosphatidylinositol (GPI) anchor attached atits C-terminus at serine 231 (Stahl et al., Cell 51:229-240 (1987)). Incommon with PrP, Dpl has a hydrophobic C-terminal region and we predictthat this may be compatible with either addition of a GPI anchor, or asingle transmembrane domain. Although Dpl lacks a serine residuecorresponding to the hamster PrP codon 231 we predict that a GPI may beattached at glycine 155. GPI attachment at a glycine has beendemonstrated for the α-folate receptor (W. Yan and M. Ratnam,Biochemistry 34:14594-600 (1995). It is interesting to note here thatexamination of rabbit PrP sequence indicates that it has no serine atresidue 231 suggesting that either rabbit PrP is not GPI anchored orthat it and may by GPI anchored by a glycine residue rather than aserine.

[0252] Conservation of residues on the surface of two evolutionarilyrelated proteins is also a useful indicator of functionally importantelements (Lichtarge et al., J. Molec. Biol. 257:342-358 (1996)). PrP hastwo consensus N-linked glycosylation sites of the form N-x-(S or T). TheN-linked glycosylation site analogous to N181 on helix B in MoPrP ismaintained in Dpl but the site on helix C is lost (FIGS. 8 and 9). Thatthis is the only conserved exposed residue on helix B argues for somefunctional significance of this complex-type glycosylation. Anadditional conserved consensus N-linked glycosylation site is found 13residues amino terminal of this position (MoDop N-72: NVT) in an exposedsurface loop. A minority of N-glycosylated proteins (˜23%) have one ormore unglycosylated consensus sites and only ˜10% of consensus sites inN-glycosylated proteins are unglycosylated (G. von Heijne, J Mol Biol.225:487-94 (1992)) indicating that this site is also likely to beglycosylated.

[0253] Electrostatic surface potential, evaluated using the GRASPalgorithm (Nicholls et al., Proteins 11:281-296 (1991)), is distinct forDpl and PrP. PrP has positively and negatively charged patches onopposing faces of the molecule (R. Riek, Nature 382:180-2), suggestingan orientation for membrane binding. Dpl maintains the positivelycharged patch seen in PrP although the boundaries of these areas are notequivalent but lacks a similar negatively charged patch (data notshown), having instead a neutral/positively-charged area. In general,the surface of Dpl has more positive charge in areas which, in the caseof PrP, are thought not to be conformationally heterogeneous (i.e.,residues 121-231). It seems likely that this disposition of chargedsurfaces might play a role in multimerization if are found to interact.Whether Dpl and PrP compete for binding to a common receptor remains tobe established.

[0254] Since PrnD mRNAs are expressed at low levels in the CNS ofwild-type animals, we sought evidence for Dpl protein in transfectedcells expressing PrnD cDNAs under the control of a constitutivecytomegalovirus promoter. Untransfected N2A cells and stably transfectedlines containing the CMV/Dpl plasmid and grown in the presence ofselectable marker were harvested for RNA, which was assessed by Northernblot analysis. Whereas 1.7 and 2.8 kb PrnD mRNAs were detected in apositive control (testis RNA), they were undetectable in untransfectedN2A cells. Of 8 stable transfected lines, 5 displayed abundant levels ofthe anticipated 0.9 kb mRNA derived from the expression plasmid.

EXAMPLE 9 Detection of Dpl by Western Blot

[0255] Two clones with abundant mRNA were selected for further analysisby western blotting, using an antiserum directed against the Dpl-2synthetic peptide. A 15 residue Dpl peptide 2 (NH-CFGAEGNRYYAANYY-COOH)corresponding to residues 71 to 84 was synthesized by FMOC chemistry andconjugated to maleimide activated KLH via a free sulfhydryl group. Theconjugated peptide was mixed with RIBI prior to subcutaneous inoculationof rabbits.

[0256] Cells were grown to approximately 80% confluency in 10 cm dishesand washed three times with 10 mL HBBS. Cells were then scraped into 5mL HBBS and pelleted at 1,000 x g for 5 min at 4 EC. The pellet wasresuspended in 0.1 mL cell lysis buffer (20 mM Tris-HCl, pH8.0, 150 mMNaCl, 0.5% Triton X-100, 0.5% sodium deoxycholate, 1 Complete miniprotease inhibitor tablet (Boehringer Mannheim)), transferred to asterile microfuge tube and agitated at 4 EC for 10 minutes. Samples werepelleted at 14,000 rpm at 4 EC for 10 minutes and supernatant wastransferred to a clean microfuge tube. Protein concentration wasdetermined by Bradford assay as recommended by the manufacturer(Bio-Rad). Samples were brought up in 4X Laemmli buffer and stored at−80 EC.

[0257] Both lines exhibited an intense heterodisperse signal with anapparent M_(r) of 30-36 KDa. A comparable signal was absent fromuntransfected cells and clones not expressing PrnD mRNA. Normalizedgel-loadings were confirmed by probing with an anti-actin antiserum.Since the predicted size of the full-length and N- and C-terminallyprocessed Dpl is 20.4 and 14.9 KDa, respectively, we infer that Dpl isheavily glycosylated. This conclusion is in accord with the presence oftwo consensus sites for N-linked glycosylation sites and offers a strongparallel to the case of PrP^(c) itself, where a 20 KDa polypeptide chainis increased in size to 33-35 KDa by the addition of carbohydratechains.

EXAMPLE 10 Rescue of Dpl Defect by Overexpression of PrP Transgene

[0258] The rescue of Ngsk Prnp^(0/0) mice by high level expression of aMoPrP-A encoded by a transgene array argues that Dpl overexpression inthese mice can be overcome by high levels of PrP^(c) (Nishida et al, inpress). In these Tg(P)Ngsk/Prnp^(0/0) mice that were rescued byoverexpression of a wild type MoPrP-A, the transgene was constructedusing the Syrian hamster Cos.Tet vector, which does not contain the DplORF (Scott et al., Protein Sci. 1:986-997 (1992)). The levels of PrnDmRNA in Tg(P)Ngsk/Prnp^(0/0) mice did not differ from Ngsk Prnp^(0/0)littermates lacking this transgene. Thus, if cerebellar degeneration isdirectly attributable to overexpression of PrnD, “rescue” byoverexpression of PrP must proceed by a postranscriptional mechanism:presumably PrP^(c) titrates-out the toxic effects of Dpl, perhaps by adirect physical interaction or competition for a common receptor.

[0259] While the present invention has been described with reference tothe specific embodiments thereof, it should be understood by thoseskilled in the art that various changes may be made and equivalents maybe substituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto. +C

What is claimed is:
 1. An isolated human doppel (Dpl) polypeptide. 2.The human Dpl polypeptide of claim 1 , comprising an amino acid sequenceof SEQ ID NO:2.
 3. An isolated nucleic acid sequence or complementthereof comprising a nucleic acid sequence encoding a human Dplpolypeptide of claim 1 .
 4. An isolated nucleic acid sequence of claim 3comprising a nucleic acid sequence of SEQ ID NO:1.
 5. A recombinantexpression vector comprising the nucleic acid sequence of claim 3 . 6.An isolated recombinant host cell containing the nucleic acid sequenceof claim 1 .
 7. A transgenic animal, comprising a genome wherein atleast one allele of an endogenous Dpl gene is altered.
 8. The transgenicanimal of claim 7 , wherein the alteration results in enhancedexpression from the Dpl gene.
 9. The transgenic animal of claim 7 ,wherein the animal further comprises a genome wherein at least oneendogenous PrP allele is ablated.
 10. The transgenic animal of claim 9 ,wherein said genome having operatively inserted therein an exogenous PrPgene from a genetically diverse species.
 11. A method for producing thehuman Dpl polypeptide of claim 1 , the method comprising the steps of:(a) culturing a recombinant host cell containing a human Dplpolypeptide-encoding nucleic acid sequence under conditions suitable forthe expression of the polypeptide; and (b) recovering the polypeptidefrom the host cell culture.
 12. An isolated antibody that specificallybinds a human Dpl polypeptide of claim 1 .
 13. The isolated antibody ofclaim 12 , wherein the antibody recognizes an epitope defined by SEQ IDNO:3.
 14. A method for identifying a nucleic acid homologous to thenucleic acid of claim 3 , the method comprising the steps of: contactinga nucleic acid probe with a test nucleic acid, the probe comprising atleast 15 contiguous nucleotides of a nucleic acid sequence encoding ahuman Dpl polypeptide; and detecting hybridization of the probe with thetest nucleic acid; wherein detection of hybridization of the probe tothe test nucleic acid indicates that the nucleic acid shares sequencehomology with the human Dpl polypeptide-encoding nucleic acid.
 15. Amethod for facilitating the detection of prion infectivity, the methodcomprising the step of: obtaining sample tissue from an animal to betested; inoculating the transgenic animal of claim 7 with the sample;and observing the transgenic animal for symptoms of prion disease. 16.The method of claim 15 , wherein the test animal is selected from thegroup consisting of human, cow, sheep, pig, horse, cat, dog, turkey orchicken, and the transgenic animal is selected from the group consistingof: mice, rats, rabbits, hamsters and guinea pigs.
 17. A method foridentifying a biologically active agent that modulates human doppel(Dpl) activity, the method comprising: combining a candidate agent withany one of: (a) a mammalian Dpl polypeptide; (b) a cell comprising anucleic acid encoding a mammalian Dpl polypeptide; (c) a cell comprisinga nucleic acid encoding a mammalian Dpl promoter sequence operablylinked to a nucleic acid encoding a report gene; or (d) a non-humantransgenic animal model comprising one of:(i) an exogenous and stablytransmitted human Dpl gene sequence; or (iii) a mammalian Dpl promotersequence operably linked to a reporter gene; and determining the effectof said agent on Dpl activity.
 18. A method for detecting in a subject apredisposition to a neurodegenerative disorder associated with a defectin doppel (Dpl) activity, the method comprising: analyzing the genomicDNA or mRNA of an individual for the presence of at least onepredisposing alteration in a genomic Dpl sequence; wherein the presenceof the altered genomic Dpl sequence is indicative of an increasedsusceptibility to a defect in neuronal outgrowth or degeneration. 19.The method of claim 17 , wherein the alteration is in a Dpl promotersequence.
 20. The method of claim 17 , wherein the alteration is in agenomic sequence encoding a Dpl polypeptide.
 21. An isolated Dpl nucleicacid, wherein said nucleic acid is comprised in part of codons from aDpl gene of a genetically diverse species.
 22. A transgenic, hybrid,non-human mammal having a genome comprised of an ablated endogenous Dplgene.
 23. The transgenic animal of claim 22 , wherein the genome furthercomprises an exogenous Dpl gene selected from the group consisting of aDpl gene from a genetically diverse species and an artificial genecomprised in part of codons from a Dpl gene of a genetically diversespecies, the hybrid mammal being characterized by being susceptible toinfection which generally only infects a genetically diverse testanimal.
 24. The hybrid mammal of claim 23 , wherein the hybrid mammalbelongs to a genus selected from the group consisting of Mus, Rattus,Oryctolagus and Mesocricetus and the test animal is selected from thegroup consisting of Bos, Ovis, Sus and Homo.
 25. A method of diagnosisof neurodegenerative disease, comprising the steps of obtaining a samplefrom a mammal suspected of having a neurodegenerative disorder; anddetecting the presence of one or more conformations of Dpl protein inthe sample.
 26. The method of claim 25 , wherein the neurodegenerativedisease is associated with Purkinje cell degeneration.